Cytokine zalpha11 ligand fusion proteins

ABSTRACT

The present invention relates to zalpha11 Ligand polynucleotide and polypeptide molecules. The zalpha11 Ligand is a novel cytokine. The polypeptides may be used within methods for stimulating the proliferation and/or development of hematopoietic cells in vitro and in vivo. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/659,684, filed Sep. 10, 2003; which is a continuation of U.S. patentapplication Ser. No. 10/295,723, filed Nov. 15, 2002, issued as U.S.Pat. No. 6,686,178 on Feb. 3, 2004 which has been filed as reissue U.S.patent application Ser. No. 11/346,580, filed Feb. 2, 2006; which areincorporated herein by reference; which is a divisional of U.S. patentapplication Ser. No. 09/923,246, filed Aug. 3, 2001, issued as U.S. Pat.No. 6,605,272 on Aug. 12, 2003: which is a divisional of U.S. patentapplication Ser. No. 09/522,217, filed Mar. 9, 2000, issued as U.S. Pat.No. 6,307,024 on Oct. 23, 2001; which claims the benefit of U.S.Provisional Application Ser. Nos. 60/123,547, filed Mar. 9, 1999;60/123,904, filed Mar. 11, 1999; and 60/142,013, filed Jul. 1, 1999.

BACKGROUND OF THE INVENTION

Proliferation and differentiation of cells of multicellular organismsare controlled by hormones and polypeptide growth factors. Thesediffusable molecules allow cells to communicate with each other and actin concert to form cells, tissues and organs, and to repair damagedtissue. Examples of hormones and growth factors include the steroidhormones (e.g. estrogen, testosterone), parathyroid hormone, folliclestimulating hormone, the interleukins, platelet derived growth factor(PDGF), epidermal growth factor (EGF), granulocyte-macrophage colonystimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding toreceptors. Receptors may be integral membrane proteins that are linkedto signaling pathways within the cell, such as second messenger systems.Other classes of receptors are soluble molecules, such as thetranscription factors.

Cytokines generally stimulate proliferation or differentiation of cellsof the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. Examples of cytokineswhich affect hematopoiesis are erythropoietin (EPO), which stimulatesthe development of red blood cells; thrombopoietin (TPO), whichstimulates development of cells of the megakaryocyte lineage; andgranulocyte-colony stimulating factor (G-CSF), which stimulatesdevelopment of neutrophils. These cytokines are useful in restoringnormal blood cell levels in patients suffering from anemia,thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.

The interleukins are a family of cytokines that mediate immunologicalresponses, including inflammation. The interleukins mediate a variety ofinflammatory pathologies. Central to an immune response is the T cell,which produce many cytokines and adaptive immunity to antigens.Cytokines produced by the T cell have been classified as type 1 and type2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998). Type 1 cytokinesinclude IL-2, IFN-γ, LT-α, and are involved in inflammatory responses,viral immunity, intracellular parasite immunity and allograft rejection.Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and areinvolved in humoral responses, helminth immunity and allergic response.Shared cytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-α.There is some evidence to suggest that Type 1 and Type 2 producing Tcell populations preferentially migrate into different types of inflamedtissue.

Mature T cells may be activated, i.e., by an antigen or other stimulus,to produce, for example, cytokines, biochemical signaling molecules, orreceptors that further influence the fate of the T cell population.

B cells can be activated via receptors on their cell surface including Bcell receptor and other accessory molecules to perform accessory cellfunctions, such as production of cytokines.

Natural killer (NK) cells have a common progenitor cell with T cells andB cells, and play a role in immune surveillance. NK cells, whichcomprise up to 15% of blood lymphocytes, do not express antigenreceptors, and therefore do not use MHC recognition as requirement forbinding to a target cell. NK cells are involved in the recognition andkilling of certain tumor cells and virally infected cells. In vivo, NKcells are believed to require activation, however, in vitro, NK cellshave been shown to kill some types of tumor cells without activation.

The demonstrated in vivo activities of the cytokine family illustratethe enormous clinical potential of, and need for, other cytokines,cytokine agonists, and cytokine antagonists. The present inventionaddresses these needs by providing a new cytokine that stimulates cellsof the hematopoietic cell lineage, as well as related compositions andmethods.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a multiple alignment of human IL-2, humanIL-15, zalpha11 Ligand (SEQ ID NO: 2), human IL-4, mouse IL-4, humanGM-CSF and mouse GM-CSF.

FIG. 2 is a Hopp/Woods hydrophilicity plot of human zalpha11 Ligand.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e. invasiveand metastatic, or benign.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a protein having the structure of afour-helical-bundle cytokine. Through processes of cloning,proliferation assays and binding studies described in detail herein, apolynucleotide sequence encoding a novel ligand polypeptide has beenidentified that is a ligand with high specificity for the previouslyorphan receptor zalpha11. This polypeptide ligand, designated zalpha11Ligand, was isolated from a cDNA library generated from activated humanperipheral blood cells (hPBCs), which were selected for CD3. CD3 is acell surface marker unique to cells of lymphoid origin, particularly Tcells.

In the examples which follow, a cell line that is dependent on thezalpha11 orphan receptor-linked pathway for survival and growth in theabsence of other growth factors was used to screen for a source of thecDNA encoding the zalpha11 Ligand. The preferred growth factor-dependentcell line that was used for transfection and expression of zalpha11receptor was BaF3 (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986). However,other growth factor-dependent cell lines, such as FDC-P1 (Hapel et al.,Blood 64: 786-790, 1984), and MO7e (Kiss et al., Leukemia 7: 235-240,1993) are suitable for this purpose.

The amino acid sequence for the zalpha11 receptor indicated that theencoded receptor belonged to the Class I cytokine receptor subfamilythat includes, but is not limited to, the receptors for IL-2, IL-4,IL-7, IL-15, EPO, TPO, GM-CSF and G-CSF (for a review see, Cosman, “TheHematopoietin Receptor Superfamily” in Cytokine 5(2): 95-106, 1993). Thezalpha11 receptor is fully described in commonly-owned PCT PatentApplication No. US99/22149. Analysis of the tissue distribution of themRNA of the zalpha11 receptor revealed expression in lymph node,peripheral blood leukocytes (PBLs), spleen, bone marrow, and thymus.Moreover, the mRNA was abundant in the Raji cell line (ATCC No. CCL-86)derived from a Burkitt's lymphoma. The tissue distribution of thereceptor suggests that a target for the predicted zalpha11 Ligand ishematopoietic lineage cells, in particular lymphoid progenitor cells andlymphoid cells. Other known four-helical-bundle cytokines that act onlymphoid cells include IL-2, IL-4, IL-7, and IL-15. For a review offour-helical-bundle cytokines, see, Nicola et al., Advances in ProteinChemistry 52:1-65, 1999 and Kelso, A., Immunol. Cell Biol. 76:300-317,1998.

Conditioned media (CM) from CD3+ selected, PMA/Ionomycin-stimulatedhuman peripheral blood cells supported the growth of BaF3 cells thatexpressed the zalpha11 receptor and were otherwise dependent on IL-3.Conditioned medias from cells that were not: 1)PMA/Ionomycin-stimulated; or were not: 2) CD3 selected (with or withoutPMA/Ionomycin stimulation) did not support the growth of BaF3/zalpha11receptor cells. Control experiments demonstrated that this proliferativeactivity was not attributable to other known growth factors, and thatthe ability of such conditioned media to stimulate proliferation ofzalpha11 receptor-expressing cells could be neutralized by a solubleform of the receptor.

Proliferation of zalpha11 receptor-expressing BaF3 cells exposed to CMfrom CD3+ selected, PMA/Ionomycin-stimulated human peripheral bloodcells were identified by visual inspection of the cultures and/or byproliferation assay. Many suitable proliferation assays are known in theart, and include assays for reduction of a dye such as alamarBlue™(AccuMed International, Inc. Westlake, Ohio),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Mosman,J. Immunol. Meth. 65: 55-63, 1983); 3,(4,5 dimethylthiazol-2yl)-5-3-carboxymethoxyphenyl-2H-tetrazolium;2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide; and cyanoditolyl-tetrazolium chloride (which are commerciallyavailable from Polysciences, Inc., Warrington, Pa.); mitogenesis assays,such as measurement of incorporation of ³H-thymidine; dye exclusionassays using, for example, naphthalene black or trypan blue; dye uptakeusing diacetyl fluorescein; and chromium release. See, in general,Freshney, Culture of Animal Cells: A Manual of Basic Technique, 3rd ed.,Wiley-Liss, 1994, which is incorporated herein by reference.

A cDNA library was prepared from CD3+ selected, PMA- andIonomycin-stimulated primary human peripheral blood cells. The CD3+selected, PMA- and Ionomycin-stimulated human peripheral blood cellscDNA library was divided into pools containing multiple cDNA moleculesand was transfected into a host cell line, for example, BHK 570 cells(ATCC accession no. 10314). The transfected host cells were cultured ina medium that did not contain exogenous growth factors and conditionedmedium was collected. The conditioned media were assayed for the abilityto stimulate proliferation of BaF3 cells transfected with the zalpha11receptor. CDNA pools producing conditioned medium that stimulatedBaF3/zalpha11 receptor cells were identified. This pooled plasmid cDNAwas electroporated into E. coli. CDNA was isolated from single coloniesand transfected individually into BHK 570 cells. Positive clones wereidentified by a positive result in the BaF3/zalpha11 receptorproliferation assay, and specificity was tested by neutralization ofproliferation using the soluble zalpha11 receptor.

A positive clone was isolated, and sequence analysis revealed that thepolynucleotide sequence contained within the plasmid DNA was novel. Thesecretory signal sequence is comprised of amino acid residues 1 (Met) to31 (Gly), and the mature polypeptide is comprised of amino acid residues32 (Gln) to 162 (Ser) (as shown in SEQ ID NO: 2).

In general, cytokines are predicted to have a four-alpha helixstructure, with helices A, C and D being most important inligand-receptor interactions, and are more highly conserved amongmembers of the family. Referring to the human zalpha11 Ligand amino acidsequence shown in SEQ ID NO:2, alignment of human zalpha11 Ligand, humanIL-15, human IL-4, and human GM-CSF amino acid sequences it is predictedthat zalpha11 Ligand helix A is defined by amino acid residues 41-56;helix B by amino acid residues 69-84; helix C by amino acid residues92-105; and helix D by amino acid residues 135-148; as shown in SEQ IDNO: 2. Structural analysis suggests that the A/B loop is long, the B/Cloop is short and the C/D loop is parallel long. This loop structureresults in an up-up-down-down helical organization. The cysteineresidues are absolutely conserved between zalpha11 Ligand and IL-15, asshown in FIG. 1. The cysteine residues that are conserved between IL-15and zalpha11 Ligand correspond to amino acid residues 71, 78, 122 and125 of SEQ ID NO: 2. Conservation of some of the cysteine residues isalso found in IL-2, IL-4, GM-CSF and zalpha11 Ligand corresponding toamino acid residues 78 and 125 of SEQ ID NO: 2, as shown in FIG. 1.Consistent cysteine placement is further confirmation of thefour-helical-bundle structure. Also highly conserved in the familycomprising IL-15, IL-2, IL-4, GM-CSF and zalpha11 Ligand is theGlu-Phe-Leu sequence as shown in SEQ ID NO: 2 at residues 136-138, as inFIG. 1.

Further analysis of zalpha11 Ligand based on multiple alignments (asshown in FIG. 1) predicts that amino acid residues 44, 47 and 135 (asshown in SEQ ID NO: 2) play an important role in zalpha11 Ligand bindingto its cognate receptor. Moreover, the predicted amino acid sequence ofmurine zalpha11 Ligand shows 57% identity to the predicted humanprotein. Based on comparison between sequences of human and murinezalpha11 Ligand well-conserved residues were found in the regionspredicted to encode alpha helices A and D. The correspondingpolynucleotides encoding the zalpha11 Ligand polypeptide regions,domains, motifs, residues and sequences described herein are as shown inSEQ ID NO: 1.

Detailed mutational analysis has been performed for IL-4 and IL-2, bothof which are highly related to zalpha11 ligand. Analysis of murine IL-2(Zurawski et al., EMBO J. 12:5113-5119, 1993) shows residues in helicesA and C are important for binding to IL-2Rβ; critical residues areAsp₃₄, Asn₉₉, and Asn₁₀₃. Multiple residues within murine IL-2 loop A/Band helix B are important for IL-2Rα binding, while only a singleresidue, Gln₁₄₁ in helix D, is vital for binding with IL-2Rα. Similarly,helices A and C are sites of interaction between IL-4 and IL-4Rα (thestructurally similar to IL-2Rα), and residues within helix D are vitalfor IL-2Rα interaction (Wang et al., Proc. Natl. Acad. Sci. USA94:1657-1662, 1997; Kruse et al., EMBO J. 11:3237-3244, 1992). Inparticular, the mutation Tyr₁₂₄ to Asp in human IL-4 creates anantagonist, which binds with IL-4Rα but not IL-2Rα and therefore cannotsignal (Kruse et al. ibid. 1992).

While helix A is relatively well-conserved between human and murinezalpha11 Ligand, helix C is more divergent. While both species havepredominant acidic amino acids in this region, the differences mayaccount for species specificity in interaction between zalpha11 Ligandand its “beta” type receptor, zalpha11 . Loop A/B and helix B ofzalpha11 Ligand are well-conserved between species; although no receptorsubunit corresponding to IL-2Rα has yet been identified, conservationthrough this region suggests that it is functionally significant. The Dhelices of human and murine zalpha11 Ligand are also highly conserved.Zalpha11 receptor antagonists may be designed through mutations withinzalpha11 Ligand helix D. These may include truncation of the proteinfrom residue Gln₁₄₅ (SEQ ID NO: 2), or mutations of Gln₁₄₅ or Ile₁₄₈ (ofSEQ ID NO: 2; corresponding to Tyr₁₂₄ in human IL-4) to residues such asAla or Asp. Any mutation which disrupts the zalpha11 Ligand helicalstructure may abolish binding with its receptor and thus inhibitsignaling.

Four-helical bundle cytokines are also grouped by the length of theircomponent helices. “Long-helix” form cytokines generally consist ofbetween 24-30 residue helices, and include IL-6, ciliary neutrotrophicfactor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone(hGH). “Short-helix” form cytokines generally consist of between 18-21residue helices and include IL-2, IL-4 and GM-CSF. Zalpha11 Ligand isbelieved to be a new member of the short-helix form cytokine group.Studies using CNTF and IL-6 demonstrated that a CNTF helix can beexchanged for the equivalent helix in IL-6, conferring CTNF-bindingproperties to the chimera. Thus, it appears that functional domains offour-helical cytokines are determined on the basis of structuralhomology, irrespective of sequence identity, and can maintain functionalintegrity in a chimera (Kallen et al., J. Biol. Chem. 274:11859-11867,1999). Therefore, the helical domains of zalpha11 Ligand will be usefulfor preparing chimeric fusion molecules, particularly with othershort-helix form cytokines to determine and modulate receptor bindingspecificity. Of particular interest are fusion proteins engineered withhelix A and/or helix D, and fusion proteins that combine helical andloop domains from other short-form cytokines such as IL-2, IL-4, IL-15and GM-CSF. The amino acid residues comprising helices A, B, C, and D,and loops A/B, B/C and C/D for zalpha11 Ligand, IL-2, IL-4, IL-15 andGM-CSF are shown in Table 1.

TABLE 1 A/B B/C C/D Helix A Loop Helix B Loop Helix C Loop Helix Dzalpha11 41-56 57-68 69-84 85-91  92-105 106-134 135-148 SEQ ID LigandNO: 2 residues IL-2 36-46 47-52 53-75 76-86  87-99 100-102 103-121 SEQID residues NO: 111 IL-4 29-43 44-64 65-83 84-94  95-118 119-133 134-151SEQ ID residues NO: 112 IL-15 45-68 69-83 84-101 102-106 107-119 120-133134-160 SEQ ID residues NO: 113 GM- 30-44 45-71 72-81 82-90  91-102103-119 120-131 SEQ ID CSF NO: 114 residues

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode the zalpha11 Ligand polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:3is a degenerate DNA sequence that encompasses all DNAs that encode thezalpha11 Ligand polypeptide of SEQ ID NO:2. Those skilled in the artwill recognize that the degenerate sequence of SEQ ID NO:3 also providesall RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus,zalpha11 Ligand polypeptide-encoding polynucleotides comprisingnucleotide 1 or 97 to nucleotide 486 of SEQ ID NO:3 and their RNAequivalents are contemplated by the present invention. Table 2 setsforth the one-letter codes used within SEQ ID NO:3 to denote degeneratenucleotide positions. “Resolutions” are the nucleotides denoted by acode letter. “Complement” indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C or T, and itscomplement R denotes A or G, with A being complementary to T, and Gbeing complementary to C.

TABLE 2 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 3.

TABLE 3 Amino One Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 3). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:3 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zalpha11 Ligand RNA. Such tissues andcells are identified by Northern blotting (Thomas, Proc. Natl. Acad.Sci. USA 77:5201, 1980), or by screening conditioned medium from variouscell types for activity on target cells or tissue. Once the activity orRNA producing cell or tissue is identified, total RNA can be preparedusing guanidinium isothiocyanate extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Nat. Acad. Sci. USA 69:1408-12, 1972).Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using knownmethods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding zalpha11 Ligand polypeptides are thenidentified and isolated by, for example, hybridization or PCR.

A full-length clone encoding zalpha11 Ligand can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones arepreferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to zalpha11 receptor fragments,or other specific binding partners.

Zalpha11 Ligand polynucleotide sequences disclosed herein can also beused as probes or primers to clone 5′ non-coding regions of a zalpha11Ligand gene. In view of the tissue-specific expression observed forzalpha11 Ligand this gene region is expected to provide forhematopoietic- and lymphoid-specific expression. Promoter elements froma zalpha11 Ligand gene could thus be used to direct the tissue-specificexpression of heterologous genes in, for example, transgenic animals orpatients treated with gene therapy. Cloning of 5′ flanking sequencesalso facilitates production of zalpha11 Ligand proteins by “geneactivation” as disclosed in U.S. Pat. No. 5,641,670. Briefly, expressionof an endogenous zalpha11 Ligand gene in a cell is altered byintroducing into the zalpha11 Ligand locus a DNA construct comprising atleast a targeting sequence, a regulatory sequence, an exon, and anunpaired splice donor site. The targeting sequence is a zalpha11 Ligand5′ non-coding sequence that permits homologous recombination of theconstruct with the endogenous zalpha11 Ligand locus, whereby thesequences within the construct become operably linked with theendogenous zalpha11 Ligand coding sequence. In this way, an endogenouszalpha11 Ligand promoter can be replaced or supplemented with otherregulatory sequences to provide enhanced, tissue-specific, or otherwiseregulated expression.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are zalpha11 Ligand polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human zalpha11 Ligand can becloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses zalpha11 Ligand as disclosed herein. Suitablesources of mRNA can be identified by probing Northern blots with probesdesigned from the sequences disclosed herein. A library is then preparedfrom mRNA of a positive tissue or cell line. A zalpha11 Ligand-encodingcDNA can then be isolated by a variety of methods, such as by probingwith a complete or partial human cDNA or with one or more sets ofdegenerate probes based on the disclosed sequences. A cDNA can also becloned using the polymerase chain reaction, or PCR (Mullis, U.S. Pat.No. 4,683,202), using primers designed from the representative humanzalpha11 Ligand sequence disclosed herein. Within an additional method,the cDNA library can be used to transform or transfect host cells, andexpression of the cDNA of interest can be detected with an antibody tozalpha11 Ligand polypeptide, binding studies or activity assays. Similartechniques can also be applied to the isolation of genomic clones.

The polynucleotide sequence for the mouse ortholog of zalpha11 Ligandhas been identified and is shown in SEQ ID NO: 55 and the correspondingamino acid sequence shown in SEQ ID NO: 56. There is 62% identitybetween the mouse and human sequences over a 124 amino acid region thatcorresponds to residues 30 to 153 in SEQ ID NO: 2 and residues 23 to 146of SEQ ID NO: 56 of zalpha11 Ligand. Mature sequence for the mousezalpha11 Ligand putatively begins at His₁₈ (as shown in SEQ ID NO: 56),which corresponds to His₂₅ (as shown in SEQ ID NO: 2) in the humansequence. Because a truncated form of the human polypeptide is active,it is likely that an equivalent polypeptide of the mouse zalpha11 Ligand(i.e. without residues His₁₈ to Pro₂₂ of SEQ ID NO: 56) is active aswell. Tissue analysis revealed that expression of mouse zalpha11 Ligandis found in testis, spleen and thymus.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human zalpha11 Ligand and thatallelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the zalpha11 Ligand polypeptide, are included within thescope of the present invention, as are polypeptides encoded by suchcDNAs and mRNAs. Allelic variants and splice variants of these sequencescan be cloned by probing cDNA or genomic libraries from differentindividuals or tissues according to standard procedures known in theart.

The zalpha11 Ligand gene has been mapped to the IL-2 framework markerSHGC-12342, positioning zalpha11 Ligand approximately 180 kb from theIL-2 marker. The use of surrounding markers positions the zalpha11Ligand gene in the 4q27 region on the integrated LDB chromosome 4 map(The Genetic Location Database, University of Southhampton,). Thepresent invention also provides reagents which will find use indiagnostic applications. For example, the zalpha11 Ligand gene, a probecomprising zalpha11 Ligand DNA or RNA or a subsequence thereof can beused to determine if the zalpha11 Ligand gene is present on a humanchromosome, such as chromosome 4, or if a gene mutation has occurred.Based on annotation of a fragment of human genomic DNA containing a partof zalpha11 Ligand genomic DNA (Genbank Accession No. AC007458),zalpha11 Ligand is located at the 4q27 region of chromosome 4.Detectable chromosomal aberrations at the zalpha11 Ligand gene locusinclude, but are not limited to, aneuploidy, gene copy number changes,loss of heterogeneity (LOH), translocations, insertions, deletions,restriction site changes and rearrangements. Such aberrations can bedetected using polynucleotides of the present invention by employingmolecular genetic techniques, such as restriction fragment lengthpolymorphism (RFLP) analysis, short tandem repeat (STR) analysisemploying PCR techniques, and other genetic linkage analysis techniquesknown in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.;Marian, Chest 108:255-65, 1995).

The precise knowledge of a gene's position can be useful for a number ofpurposes, including: 1) determining if a sequence is part of an existingcontig and obtaining additional surrounding genetic sequences in variousforms, such as YACs, BACs or cDNA clones; 2) providing a possiblecandidate gene for an inheritable disease which shows linkage to thesame chromosomal region; and 3) cross-referencing model organisms, suchas mouse, which may aid in determining what function a particular genemight have.

As stated previously, human zalpha11 Ligand gene resides near the IL-2gene, which is in a region of chromosome 4q that has been shown to havelinkage with susceptibility to inflammatory bowel disease (IBD)(including Crohn's disease (CD) and ulcerative colitis) in some families(Hampe et al. Am. J. Hum. Genet. 64:808-816, 1999; Cho et al. Proc.Natl. Acad. Sci. 95:7502-7507, 1998). In addition, the zalpha11 receptorgene maps to 16p11, another genomic region which is associated withsusceptibility to CD (Hugot et al., Nature 379:821-823, 1996; Ohmen etal., Hum. Mol. Genet. 5:1679-1683, 1996). CD is a chronic inflammationof the gut with frequent systemic involvement; while the exact etiologyis unknown, immunoregulatory dysfunction involving failure of toleranceto ordinary gut antigens is a major component (for reviews, see(Braegger et al., Annals Allergy 72:135-141, 1994; Sartor, Am. J.Gastroenterol. 92:5S11S, 1997)). Several studies have found abnormal NKactivity in CD patients (see, for example, (Egawa et. al., J. Clin. Lab.Immunol. 20:187-192, 1986; Aparicio-Pagés et al. J. Clin. Lab. Immunol.29:119-124, 1989; van Tol et al., Scand. J. Gastroenterol. 27:999-1005,1992)), and defective memory B cell formation has also been documented(Brogan et al., J. Clin. Lab. Immunol. 24:69-74, 1987). Since zalpha11Ligand plays a role in immune regulation, and since the genes for bothreceptor and ligand lie within CD susceptibility regions, both receptorand ligand are candidate genes for genetic predisposition to Crohn'sdisease.

Determination of the involvement of zalpha11 receptor and/or zalpha11Ligand in the pathology of IBD can be accomplished by several methods.Sequencing of exons from genomic DNA can reveal coding mutations(including missense, nonsense, and frameshift mutations), as cansequencing of cDNAs. An additional advantage of sequencing from genomicDNA is that splice junctions are also contained within the sequencedfragments and may reveal splicing abnormalities, which might not appearin cDNA samples if, for example, misspliced RNAs were rapidly degraded.The genomic structure of zalpha11 Ligand has been determined. Othermethods for analysis of zalpha11 Ligand and receptor in IBD patientsinclude: (1) assessment of ligand production from activated T cells frompatients vs. normal controls (i.e. by bioassay); (2) in situhybridization of zalpha11 receptor or zalpha11 Ligand RNA to sections ofinflamed intestine from IBD patients, compared to similar sections fromnormal controls; (3) immunohistochemistry on sections from IBD patientsvs. normal controls; and (4) assessment of the responsiveness ofpatients' peripheral B cells to zalpha11 Ligand, as measured bymitogenesis assays.

A diagnostic could assist physicians in determining the type of diseaseand appropriate associated therapy, or could assist in geneticcounseling. As such, the inventive anti-zalpha11 Ligand antibodies,polynucleotides, and polypeptides can be used for the detection ofzalpha11 Ligand polypeptide, mRNA or anti-zalpha11 Ligand antibodies,thus serving as markers and be directly used for detecting or geneticdiseases or cancers, as described herein, using methods known in the artand described herein. Further, zalpha11 Ligand polynucleotide probes canbe used to detect abnormalities involving chromosome 4q27 as describedherein. These abnormalities may be associated with human diseases, ortumorigenesis, spontaneous abortion or other genetic disorders. Thus,zalpha11 Ligand polynucleotide probes can be used to detectabnormalities or genotypes associated with these defects.

As discussed above, defects in the zalpha11 Ligand gene itself mayresult in a heritable human disease state. Molecules of the presentinvention, such as the polypeptides, antagonists, agonists,polynucleotides and antibodies of the present invention would aid in thedetection, diagnosis prevention, and treatment of diseases associatedwith a zalpha11 Ligand genetic defect. In addition, zalpha11 Ligandpolynucleotide probes can be used to detect allelic differences betweendiseased or non-diseased individuals at the zalpha11 Ligand chromosomallocus. As such, the zalpha11 Ligand sequences can be used as diagnosticsin forensic DNA profiling.

In general, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Most diagnostic methods comprise the steps of (i) obtaining agenetic sample from a potentially diseased patient, diseased patient orpotential non-diseased carrier of a recessive disease allele; (ii)producing a first reaction product by incubating the genetic sample witha zalpha11 Ligand polynucleotide probe wherein the polynucleotide willhybridize to complementary polynucleotide sequence, such as in RFLPanalysis or by incubating the genetic sample with sense and antisenseprimers in a PCR reaction under appropriate PCR reaction conditions;(iii) Visualizing the first reaction product by gel electrophoresisand/or other known method such as visualizing the first reaction productwith a zalpha11 Ligand polynucleotide probe wherein the polynucleotidewill hybridize to the complementary polynucleotide sequence of the firstreaction; and (iv) comparing the visualized first reaction product to asecond control reaction product of a genetic sample from a normal orcontrol individual. A difference between the first reaction product andthe control reaction product is indicative of a genetic abnormality inthe diseased or potentially diseased patient, or the presence of aheterozygous recessive carrier phenotype for a non-diseased patient, orthe presence of a genetic defect in a tumor from a diseased patient, orthe presence of a genetic abnormality in a fetus or pre-implantationembryo. For example, a difference in restriction fragment pattern,length of PCR products, length of repetitive sequences at the zalpha11Ligand genetic locus, and the like, are indicative of a geneticabnormality, genetic aberration, or allelic difference in comparison tothe normal control. Controls can be from unaffected family members, orunrelated individuals, depending on the test and availability ofsamples. Genetic samples for use within the present invention includegenomic DNA, mRNA, and cDNA isolated fromm any tissue or otherbiological sample from a patient, such as but not limited to, blood,saliva, semen, embryonic cells, amniotic fluid, and the like. Thepolynucleotide probe or primer can be RNA or DNA, and will comprise aportion of SEQ ID NO:1, the complement of SEQ ID NO:1, or an RNAequivalent thereof. Such methods of showing genetic linkage analysis tohuman disease phenotypes are well known in the art. For reference to PCRbased methods in diagnostics see, generally, Mathew (ed.), Protocols inHuman Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCRProtocols: Current Methods and Applications (Humana Press, Inc. 1993),Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996),Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc.1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998),and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

Mutations associated with the zalpha11 Ligand locus can be detectedusing nucleic acid molecules of the present invention by employingstandard methods for direct mutation analysis, such as restrictionfragment length polymorphism analysis, short tandem repeat analysisemploying PCR techniques, amplification-refractory mutation systemanalysis, single-strand conformation polymorphism detection, RNasecleavage methods, denaturing gradient gel electrophoresis,fluorescence-assisted mismatch analysis, and other genetic analysistechniques known in the art (see, for example, Mathew (ed.), Protocolsin Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (HumanPress, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases(Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols forMutation Detection (Oxford University Press 1996), Birren et al. (eds.),Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor LaboratoryPress 1998), Dracopoli et al. (eds.), Current Protocols in HumanGenetics (John Wiley & Sons 1998), and Richards and Ward, “MolecularDiagnostic Testing,” in Principles of Molecular Medicine, pages 83-88(Humana Press, Inc. 1998). Direct analysis of an zalpha11 Ligand genefor a mutation can be performed using a subject's genomic DNA. Methodsfor amplifying genomic DNA, obtained for example from peripheral bloodlymphocytes, are well-known to those of skill in the art (see, forexample, Dracopoli et al. (eds.), Current Protocols in Human Genetics,at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).

Positions of introns in the zalpha11 Ligand gene were determined byidentification of genomic clones, followed by sequencing the intron/exonjunctions. The first intron lies between amino acid residue 56 (Leu) andresidue 57 (Val) in Seq. ID. No. 2, and is 115 base pairs in length. Thesecond intron is the largest at 4.4 kilobases, and lies between aminoacid residue 68 (Glu) and residue 69 (Thr) in Seq. ID. No. 2. The thirdintron is 2.6 kilobases, and lies between amino acid residue 120 (Leu)and residue 121 (Thr) in Seq. ID. No. 2. The final intron, 89 basepairs, lies between amino acid residue 146 (Lys) and residue 147 (Met)in Seq. ID. No. 2. The complete gene spans about 8 kb.

The structure of the zalpha11 Ligand gene is similar to that of the IL-2gene (Fujita et al. Proc. Natl. Acad. Sci. 80:7437-7441, 1983), thoughthe zalpha11 Ligand gene contains one additional intron (intron 4). Thepattern of a short first intron and long second and third introns isconserved between the two genes, though the IL-2 gene is slightlysmaller overall (about 6 kb). The IL-15 gene, on the other hand,consists of 8 exons and spans at least 34 kb (Anderson et al. Genomics25:701-706, 1995). Thus the zalpha11 Ligand gene is more similar instructure to the IL-2 gene than to the IL-15 gene.

Within embodiments of the invention, isolated zalpha11 Ligand-encodingnucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules having the nucleotide sequence of SEQ ID NO:1, tonucleic acid molecules having the nucleotide sequence of nucleotides 47to 532 of SEQ ID NO: 1, or to nucleic acid molecules having a nucleotidesequence complementary to SEQ ID NO:1. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases.

It is well within the abilities of one skilled in the art to adapt theseconditions for use with a particular polynucleotide hybrid. The T_(m)for a, specific target sequence is the temperature (under definedconditions) at which 50% of the target sequence will hybridize to aperfectly matched probe sequence. Those conditions which influence theT_(m) include, the size and base pair content of the polynucleotideprobe, the ionic strength of the hybridization solution, and thepresence of destabilizing agents in the hybridization solution. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding a variant zalpha11 Ligand polypeptide hybridize with a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1 (or itscomplement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.,including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantzalpha11 Ligand polypeptide hybridize with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO: 1 (or its complement) underhighly stringent washing conditions, in which the wash stringency isequivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSCwith 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

The present invention also provides isolated zalpha11 Ligandpolypeptides that have a substantially similar sequence identity to thepolypeptides of SEQ ID NO:2, or their orthologs. The term “substantiallysimilar sequence identity” is used herein to denote polypeptidescomprising at least 70%, at least 80%, at least 90%, at least 95%, orgreater than 95% sequence identity to the sequences shown in SEQ IDNO:2, or their orthologs. The present invention also includespolypeptides that comprise an amino acid sequence having at least 70%,at least 80%, at least 90%, at least 95% or greater than 95% sequenceidentity to the sequence of amino acid residues 1 to 162 or 32 to 162 ofSEQ ID NO:2. The present invention further includes nucleic acidmolecules that encode such polypeptides. Methods for determining percentidentity are described below.

The present invention also contemplates variant zalpha11 Ligand nucleicacid molecules that can be identified using two criteria: adetermination of the similarity between the encoded polypeptide with theamino acid sequence of SEQ ID NO:2, and/or a hybridization assay, asdescribed above. Such zalpha11 Ligand variants include nucleic acidmolecules: (1) that hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under stringentwashing conditions, in which the wash stringency is equivalent to0.5×-2×SSC with 0.1% SDS at 55-65° C.; or (2) that encode a polypeptidehaving at least 70%, at least 80%, at least 90%, at least 95% or greaterthan 95% sequence identity to the amino acid sequence of SEQ ID NO:2.Alternatively, zalpha11 Ligand variants can be characterized as nucleicacid molecules: (1) that hybridize with a nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO: 1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.; and (2) that encode apolypeptide having at least 70%, at least 80%, at least 90%, at least95% or greater than 95% sequence identity to the amino acid sequence ofSEQ ID NO:2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 4 (amino acids are indicated by the standard one-lettercodes).

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{matrix}\begin{matrix}\lbrack {{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{20mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}}\mspace{14mu}  \\{{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}}\mspace{14mu}}\end{matrix} \\ {{sequence}\mspace{20mu}{in}\mspace{14mu}{order}\mspace{20mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}} \rbrack\end{matrix}} \times 100$

TABLE 4 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant zalpha11 Ligand. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

Variant zalpha11 Ligand polypeptides or polypeptides with substantiallysimilar sequence identity are characterized as having one or more aminoacid substitutions, deletions or additions. These changes are preferablyof a minor nature, that is conservative amino acid substitutions (seeTable 5) and other substitutions that do not significantly affect thefolding or activity of the polypeptide; small deletions, typically ofone to about 30 amino acids; and amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides of from about 108 to 216 amino acid residuesthat comprise a sequence that is at least 70%, preferably at least 90%,and more preferably 95% or more identical to the corresponding region ofSEQ ID NO:2. Polypeptides comprising affinity tags can further comprisea proteolytic cleavage site between the zalpha11 Ligand polypeptide andthe affinity tag. Preferred such sites include thrombin cleavage sitesand factor Xa cleavage sites.

TABLE 5 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in zalpha11 Ligand polypeptides soas to minimize disruption of higher order structure essential tobiological activity. For example, where the zalpha11 Ligand polypeptidecomprises one or more helices, changes in amino acid residues will bemade so as not to disrupt the helix geometry and other components of themolecule where changes in conformation abate some critical function, forexample, binding of the molecule to its binding partners, e.g., A and Dhelices, residues 44, 47 and 135 of SEQ ID NO: 2. The effects of aminoacid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same cysteine pattern as thestandard molecule folding would be affected. Another well known andaccepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructurally similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the zalpha11 Ligand proteinsequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc.Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986and Triquier et al., Protein Engineering 11:153-169, 1998). The profileis based on a sliding six-residue window. Buried G, S, and T residuesand exposed H, Y, and W residues were ignored. For example, in zalpha11Ligand, hydrophilic regions include amino acid residues 114-119 of SEQID NO: 2, amino acid residues 101-105 of SEQ ID NO: 2, amino acidresidues 126-131 of SEQ ID NO: 2, amino acid residues 113-118 of SEQ IDNO: 2, and amino acid residues 158-162 of SEQ ID NO: 2.

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zalpha11 Ligand polypeptide, so as notto disrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include residues 100 and 103 as shown in SEQ ID NO:2. Cysteine residues at positions 71, 78, 122 and 125 of SEQ ID NO: 2,will be relatively intolerant of substitution.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between IL-15, IL-2, IL-4 and GM-CSFwith zalpha11 Ligand. Using methods such as “FASTA” analysis describedpreviously, regions of high similarity are identified within a family ofproteins and used to analyze amino acid sequence for conserved regions.An alternative approach to identifying a variant zalpha11 Ligandpolynucleotide on the basis of structure is to determine whether anucleic acid molecule encoding a potential variant zalpha11 Ligand genecan hybridize to a nucleic acid molecule having the nucleotide sequenceof SEQ ID NO:1, as discussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalor biochemical activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699 (1996).

The present invention also includes functional fragments of zalpha11Ligand polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” zalpha11 Ligand or fragment thereof as definedherein is characterized by its proliferative or differentiatingactivity, by its ability to induce or inhibit specialized cellfunctions, or by its ability to bind specifically to an anti-zalpha11Ligand antibody or zalpha11 receptor (either soluble or immobilized). Aspreviously described herein, zalpha11 Ligand is characterized by afour-helical-bundle structure comprising helix A (amino acid residues41-56), helix B (amino acid residues 69-84), helix C (amino acidresidues 92-105) and helix D (amino acid residues 135-148), as shown inSEQ ID NO: 2. Thus, the present invention further provides fusionproteins encompassing: (a) polypeptide molecules comprising one or moreof the helices described above; and (b) functional fragments comprisingone or more of these helices. The other polypeptide portion of thefusion protein may be contributed by another four-helical-bundlecytokine, such as IL-15, IL-2, IL-4 and GM-CSF, or by a non-nativeand/or an unrelated secretory signal peptide that facilitates secretionof the fusion protein.

Thus the present invention provides fusion proteins comprising at leastfour polypeptides, wherein the order of polypeptides from N-terminus toC-terminus are: a first polypeptide comprises amino acids selected froma group consisting of: (a) IL-2 helix A amino acid residues 36-46 of SEQID NO: 111; (b) IL-15 helix A amino acid residues 29-43 of SEQ ID NO:112; (c) IL4 helix A amino acid residues 45-68 of SEQ ID NO: 113; (d)GMCSF helix A amino acid residues 30-44 of SEQ ID NO: 114; and (e) aminoacids residues 41 to 56 of SEQ ID NO: 2; a first spacer of 6-27 aminoacids; and a second polypeptide that comprises amino acid residuesselected from the group consisting of: (a) IL-2 helix B amino acidresidues 53-75 of SEQ ID NO: 111; (b) IL-4 helix B amino acid residues65-83 of SEQ ID NO: 112; (c) IL-15 helix B amino acid residues 84-101 ofSEQ ID NO: 113; (d) GMCSF helix B amino acid residues 72-81 of SEQ IDNO: 114; and (e) amino acid residues 69-84 of SEQ ID NO: 2; a secondspacer of 5-11 amino acid residues; a third polypeptide that comprises asequence of amino acid residues selected from the group consisting of:(a) IL-2 helix C residues 87-99 of SEQ ID NO: 111; (b) IL-4 helix Cresidues 95-118 of SEQ ID NO: 112; (c) IL-15 helix C residues 107-119 ofSEQ ID NO: 113; (d) GMCSF helix C residues 91-102 of SEQ ID NO: 114; and(e) amino acid residues 92-105 of SEQ ID NO: 2; a third spacer of 3-29amino acid residues; and a fourth polypeptide that comprises amino acidresidues selected from the group consisting of: (a) IL-2 helix D aminoacid residues 103-121 of SEQ ID NO: 111; (b) IL-15 helix D amino acidresidues 134-157 of SEQ ID NO: 112; (c) IL-4 helix D amino acid residues134-160 of SEQ ID NO: 113; (d) GMCSF helix D amino acid residues 120-131of SEQ ID NO: 114; and (e) amino acid residues 135-148 of SEQ ID NO: 2,wherein at least one of the four polypeptides is from zalpha11 Ligand.In other embodiments that the spacer peptides will be selected from theA/B, B/C and C/D loops of zalpha11 Ligand, IL-2, IL-4, IL-15 or GM-CSF,as shown in Table 1.

Routine deletion analyses of nucleic acid molecules can be performed toobtain functional fragments of a nucleic acid molecule that encodes azalpha11 Ligand polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO: 1 or fragments thereof, can bedigested with Bal31nuclease to obtain a series of nested deletions.These DNA fragments are then inserted into expression vectors in properreading frame, and the expressed polypeptides are isolated and testedfor zalpha11 Ligand activity, or for the ability to bind anti-zalpha11Ligand antibodies or zalpha11 receptor. One alternative to exonucleasedigestion is to use oligonucleotide-directed mutagenesis to introducedeletions or stop codons to specify production of a desired zalpha11Ligand fragment. Alternatively, particular fragments of a zalpha11Ligand gene can be synthesized using the polymerase chain reaction.

Standard methods for identifying functional domains are well-known tothose of skill in the art. For example, studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1, Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204), and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed zalpha11 Ligand nucleotide and polypeptidesequences can also be generated through DNA shuffling as disclosed byStemmer, Nature 370:389 (1994), Stemmer, Proc. Natl Acad. Sci. USA91:10747 (1994), and international publication No. WO 97/20078. Briefly,variant DNA molecules are generated by in vitro homologous recombinationby random fragmentation of a parent DNA followed by reassembly usingPCR, resulting in randomly introduced point mutations. This techniquecan be modified by using a family of parent DNA molecules, such asallelic variants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-zalpha11 Ligand antibodies or soluble zalpha11receptor, can be recovered from the host cells and rapidly sequencedusing modern equipment. These methods allow the rapid determination ofthe importance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

In addition, the proteins of the present invention (or polypeptidefragments thereof) can be joined to other bioactive molecules,particularly other cytokines, to provide multi-functional molecules. Forexample, one or more helices from zalpha11 Ligand can be joined to othercytokines to enhance their biological properties or efficiency ofproduction.

The present invention thus provides a series of novel, hybrid moleculesin which a segment comprising one or more of the helices of zalpha11Ligand is fused to another polypeptide. Fusion is preferably done bysplicing at the DNA level to allow expression of chimeric molecules inrecombinant production systems. The resultant molecules are then assayedfor such properties as improved solubility, improved stability,prolonged clearance half-life, improved expression and secretion levels,and pharmacodynamics. Such hybrid molecules may further compriseadditional amino acid residues (e.g. a polypeptide linker) between thecomponent proteins or polypeptides.

Non-naturally occurring amino acids include, without limitation,trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,trans-4-hydroxyproline, N-methylglycine, allo-threonine,methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylicacid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is typically carried out in acell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993). It may be advantageous tostabilize zalpha11 Ligand to extend the half-life of the molecule,particularly for extending metabolic persistence in an active state. Toachieve extended half-life, zalpha11 Ligand molecules can be chemicallymodified using methods described herein. PEGylation is one methodcommonly used that has been demonstrated to increase plasma half-life,increased solubility, and decreased antigenicity and immunogenicity(Nucci et al., Advanced Drug Delivery Reviews 6:133-155, 1991 and Lu etal., Int. J. Peptide Protein Res. 43:127-138, 1994).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zalpha11 Ligand aminoacid residues.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a zalpha11 Ligand polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein. Hopp/Woodshydrophilicity profiles can be used to determine regions that have themost antigenic potential (Hopp et al., 1981, ibid. and Hopp, 1986,ibid.). In zalpha11 Ligand these regions include: amino acid residues114-119, 101-105, 126-131, 113-118, and 158-162 of SEQ ID NO: 2.

Antigenic epitope-bearing peptides and polypeptides preferably containat least four to ten amino acids, at least ten to fourteen amino acids,or about fourteen to about thirty amino acids of SEQ ID NO:2 or SEQ IDNO:56. Such epitope-bearing peptides and polypeptides can be produced byfragmenting a zalpha11 Ligand polypeptide, or by chemical peptidesynthesis, as described herein. Moreover, epitopes can be selected byphage display of random peptide libraries (see, for example, Lane andStephen, Curr. Opin. Immunol. 5:268 (1993); and Cortese et al., Curr.Opin. Biotechnol. 7:616 (1996)). Standard methods for identifyingepitopes and producing antibodies from small peptides that comprise anepitope are described, for example, by Mole, “Epitope Mapping,” inMethods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (TheHumana Press, Inc. 1992); Price, “Production and Characterization ofSynthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies:Production, Engineering, and Clinical Application, Ritter and Ladyman(eds.), pages 60-84 (Cambridge University Press 1995), and Coligan etal. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

Regardless of the particular nucleotide sequence of a variant zalpha11Ligand polynucleotide, the polynucleotide encodes a polypeptide that ischaracterized by its proliferative or differentiating activity, itsability to induce or inhibit specialized cell functions, or by theability to bind specifically to an anti-zalpha11 Ligand antibody orzalpha11 receptor. More specifically, variant zalpha11 Ligandpolynucleotides will encode polypeptides which exhibit at least 50% andpreferably, greater than 70%, 80% or 90%, of the activity of thepolypeptide as shown in SEQ ID NO: 2.

For any zalpha11 Ligand polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2 above.

The present invention further provides a variety of other polypeptidefusions (and related multimeric proteins comprising one or morepolypeptide fusions). For example, a zalpha11 Ligand polypeptide can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains.Immunoglobulin-zalpha11 Ligand polypeptide fusions can be expressed ingenetically engineered cells (to produce a variety of multimericzalpha11 Ligand analogs). Auxiliary domains can be fused to zalpha11Ligand polypeptides to target them to specific cells, tissues, ormacromolecules. For example, a zalpha11 Ligand polypeptide or proteincould be targeted to a predetermined cell type by fusing a zalpha11Ligand polypeptide to a ligand that specifically binds to a receptor onthe surface of that target cell. In this way, polypeptides and proteinscan be targeted for therapeutic or diagnostic purposes. A zalpha11Ligand polypeptide can be fused to two or more moieties, such as anaffinity tag for purification and a targeting domain. Polypeptidefusions can also comprise one or more cleavage sites, particularlybetween domains. See, Tuan et al., Connective Tissue Research 34:1-9,1996.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that havesubstantially similar sequence identity to residues 1-162 or 32-162 ofSEQ ID NO: 2, or functional fragments and fusions thereof, wherein suchpolypeptides or fragments or fusions retain the properties of thewild-type protein such as the ability to stimulate proliferation,differentiation, induce specialized cell function or bind the zalpha11receptor or zalpha11 Ligand antibodies.

The zalpha11 Ligand polypeptides of the present invention, includingfull-length polypeptides, functional fragments, and fusion polypeptides,can be produced in genetically engineered host cells according toconventional techniques. Suitable host cells are those cell types thatcan be transformed or transfected with exogenous DNA and grown inculture, and include bacteria, fungal cells, and cultured highereukaryotic cells. Eukaryotic cells, particularly cultured cells ofmulticellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987.

In general, a DNA sequence encoding a zalpha11 Ligand polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

To direct a zalpha11 Ligand polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zalpha11 Ligand, or may bederived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is operably linked to the zalpha11Ligand DNA sequence, i.e., the two sequences are joined in the correctreading frame and positioned to direct the newly synthesized polypeptideinto the secretory pathway of the host cell. Secretory signal sequencesare commonly positioned 5′ to the DNA sequence encoding the polypeptideof interest, although certain secretory signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid residue 1-31of SEQ ID NO:2 is be operably linked to a DNA sequence encoding anotherpolypeptide using methods known in the art and disclosed herein. Thesecretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Such constructs have numerous applications known in the art. Forexample, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used in vivo or in vitro to direct peptidesthrough the secretory pathway.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. The second method of making recombinant baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Backit (Life Technologies, Rockville, Md.). This system utilizes a transfervector, pFastBac1™ (Life Technologies) containing a Tn7 transposon tomove the DNA encoding the zalpha11 Ligand polypeptide into a baculovirusgenome maintained in E. coli as a large plasmid called a “bacmid.” ThepFastBac1™ transfer vector utilizes the AcNPV polyhedrin promoter todrive the expression of the gene of interest, in this case zalpha11Ligand. However, pFastBac1™ can be modified to a considerable degree.The polyhedrin promoter can be removed and substituted with thebaculovirus basic protein promoter (also known as Pcor, p6.9 or MPpromoter) which is expressed earlier in the baculovirus infection, andhas been shown to be advantageous for expressing secreted proteins. See,Hill-Perkins, M. S. and Possee, R. D., J. Gen. Virol. 71:971-6, 1990;Bonning, B. C. et al., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk,G. D., and Rapoport, B., J. Biol. Chem. 270:1543-9, 1995. In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native zalpha11 Ligand secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67(PharMingen, San Diego, Calif.) can be used in constructs to replace thenative zalpha11 Ligand secretory signal sequence. In addition, transfervectors can include an in-frame fusion with DNA encoding an epitope tagat the C- or N-terminus of the expressed zalpha11 Ligand polypeptide,for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl.Acad. Sci. 82:7952-4, 1985). Using techniques known in the art, atransfer vector containing zalpha11 Ligand is transformed into E. Coli,and screened for bacmids which contain an interrupted lacZ geneindicative of recombinant baculovirus. The bacmid DNA containing therecombinant baculovirus genome is isolated, using common techniques, andused to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.Recombinant virus that expresses zalpha11 Ligand is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (Ω) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zalpha11Ligand polypeptide in bacteria such as E. coli, the polypeptide may beretained in the cytoplasm, typically as insoluble granules, or may bedirected to the periplasmic space by a bacterial secretion sequence. Inthe former case, the cells are lysed, and the granules are recovered anddenatured using, for example, guanidine isothiocyanate or urea. Thedenatured polypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

It is preferred to purify the polypeptides of the present invention to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified polypeptideis substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Expressed recombinant zalpha11 Ligand polypeptides (or chimeric zalpha11Ligand polypeptides) can be purified using fractionation and/orconventional purification methods and media. Ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their physical or biochemical properties. For example,immobilized metal ion adsorption (IMAC) chromatography can be used topurify histidine-rich proteins, including those comprising polyhistidinetags. Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39) and use of the solublezalpha11 receptor. Within additional embodiments of the invention, afusion of the polypeptide of interest and an affinity tag (e.g.,maltose-binding protein, an immunoglobulin domain) may be constructed tofacilitate purification.

Moreover, using methods described in the art, polypeptide fusions, orhybrid zalpha11 Ligand proteins, are constructed using regions ordomains of the inventive zalpha11 Ligand in combination with those ofother human cytokine family proteins (e.g. interleukins or GM-CSF), orheterologous proteins (Sambrook et al., ibid., Altschul et al., ibid.,Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein).These methods allow the determination of the biological importance oflarger domains or regions in a polypeptide of interest. Such hybrids mayalter reaction kinetics, binding, constrict or expand the substratespecificity, or alter tissue and cellular localization of a polypeptide,and can be applied to polypeptides of unknown structure.

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. For example, part or all of a helix conferring a biologicalfunction may be swapped between zalpha11 Ligand of the present inventionwith the functionally equivalent helices from another family member,such as IL-15, IL-2, IL-4 or GM-CSF. Such components include, but arenot limited to, the secretory signal sequence; helices A, B, C, D; loopsA/B, B/C, C/D; of four-helical-bundle cytokines. Such fusion proteinswould be expected to have a biological functional profile that is thesame or similar to polypeptides of the present invention or other knownfour-helical-bundle cytokine family proteins, depending on the fusionconstructed. Moreover, such fusion proteins may exhibit other propertiesas disclosed herein.

Standard molecular biological and cloning techniques can be used to swapthe equivalent domains between the zalpha11 Ligand polypeptide and thosepolypeptides to which they are fused. Generally, a DNA segment thatencodes a domain of interest, e.g., zalpha11 Ligand helices A through D,or other domain described herein, is operably linked in frame to atleast one other DNA segment encoding an additional polypeptide (forinstance a domain or region from another cytokine, such as the IL-2, orthe like), and inserted into an appropriate expression vector, asdescribed herein. Generally DNA constructs are made such that theseveral DNA segments that encode the corresponding regions of apolypeptide are operably linked in frame to make a single construct thatencodes the entire fusion protein, or a functional portion thereof. Forexample, a DNA construct would encode from N-terminus to C-terminus afusion protein comprising a signal polypeptide followed by a mature fourhelical bundle cytokine fusion protein containing helix A, followed byhelix B, followed by helix C, followed by helix D. Such fusion proteinscan be expressed, isolated, and assayed for activity as describedherein.

Zalpha11 Ligand polypeptides or fragments thereof may also be preparedthrough chemical synthesis. zalpha11 Ligand polypeptides may be monomersor multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue. For example, the polypeptides can be prepared by solidphase peptide synthesis, for example as described by Merrifield, J. Am.Chem. Soc. 85:2149, 1963.

The activity of molecules of the present invention can be measured usinga variety of assays that measure proliferation of and/or binding tocells expressing the zalpha11 receptor. Of particular interest arechanges in zalpha11 Ligand-dependent cells. Suitable cell lines to beengineered to be zalpha11 Ligand-dependent include the IL-3-dependentBaF3 cell line (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), FDC-PI(Hapel et al., Blood 64: 786-790, 1984), and MO7e (Kiss et al., Leukemia7: 235-240, 1993). Growth factor-dependent cell lines can be establishedaccording to published methods (e.g. Greenberger et al., Leukemia Res.8: 363-375, 1984; Dexter et al., in Baum et al. Eds., ExperimentalHematology Today, 8th Ann. Mtg. Int. Soc. Exp. Hematol. 1979, 145-156,1980).

Proteins of the present invention are useful for stimulatingproliferation, activation, differentiation and/or induction orinhibition of specialized cell function of cells of the involvedhomeostasis of the hematopoiesis and immune function. In particular,zalpha11 Ligand polypeptides are useful for stimulating proliferation,activation, differentiation, induction or inhibition of specialized cellfunctions of cells of the hematopoietic lineages, including, but notlimited to, T cells, B cells, NK cells, dendritic cells, monocytes, andmacrophages, as well as epithelial cells. Proliferation and/ordifferentiation of hematopoietic cells can be measured in vitro usingcultured cells or in vivo by administering molecules of the claimedinvention to the appropriate animal model. Assays measuring cellproliferation or differentiation are well known in the art. For example,assays measuring proliferation include such assays as chemosensitivityto neutral red dye (Cavanaugh et al., Investigational New Drugs8:347-354, 1990, incorporated herein by reference), incorporation ofradiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7,1989, incorporated herein by reference), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporatedherein by reference), and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., CancerRes. 48:4827-4833, 1988; all incorporated herein by reference). Assaysmeasuring differentiation include, for example, measuring cell-surfacemarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference).

The molecules of the present invention can be assayed in vivo usingviral delivery systems. Exemplary viruses for this purpose includeadenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997).

As a ligand, the activity of zalpha11 Ligand polypeptide can be measuredby a silicon-based biosensor microphysiometer which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method. See, for example, McConnell, H. M. etal., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95, 1998.

Moreover, zalpha11 Ligand can be used to identify cells, tissues, orcell lines which respond to a zalpha11 Ligand-stimulated pathway. Themicrophysiometer, described above, can be used to rapidly identifyligand-responsive cells, such as cells responsive to zalpha11 Ligand ofthe present invention. Cells can be cultured in the presence or absenceof zalpha11 Ligand polypeptide. Those cells which elicit a measurablechange in extracellular acidification in the presence of zalpha11 Ligandare responsive to zalpha11 Ligand. Such cells or cell lines, can be usedto identify antagonists and agonists of zalpha11 Ligand polypeptide asdescribed above.

In view of the tissue distribution observed for zalpha11 receptoragonists (including the natural zalpha11 Ligand/substrate/cofactor/etc.)and/or antagonists have enormous potential in both in vitro and in vivoapplications. Compounds identified as zalpha11 Ligand agonists areuseful for expansion, proliferation, activation, differentiation, and/orinduction or inhibition of specialized cell functions of cells involvedin homeostasis of hematopoiesis and immune function. For example,zalpha11 Ligand and agonist compounds are useful as components ofdefined cell culture media, and may be used alone or in combination withother cytokines and hormones to replace serum that is commonly used incell culture. Agonists are thus useful in specifically promoting thegrowth and/or development of T-cells, B-cells, NK cells, cytotoxiclymphocytes, and other cells of the lymphoid and myeloid lineages inculture.

Antagonists are also useful as research reagents for characterizingsites of ligand-receptor interaction. Antagonists are useful to inhibitexpansion, proliferation, activation, and/or differentiation of cellsinvolved in regulating hematopoiesis. Inhibitors of zalpha11 Ligandactivity (zalpha11 Ligand antagonists) include anti-zalpha11 Ligandantibodies and soluble zalpha11 Ligand receptors, as well as otherpeptidic and non-peptidic agents (including ribozymes). zalpha11 Ligandcan also be used to identify inhibitors (antagonists) of its activity.Test compounds are added to the assays disclosed herein to identifycompounds that inhibit the activity of zalpha11 Ligand. In addition tothose assays disclosed herein, samples can be tested for inhibition ofzalpha11 Ligand activity within a variety of assays designed to measurereceptor binding, the stimulation/inhibition of zalpha11Ligand-dependent cellular responses or proliferation of zalpha11receptor-expressing cells.

A zalpha11 Ligand polypeptide can be expressed as a fusion with animmunoglobulin heavy chain constant region, typically an F_(C) fragment,which contains two constant region domains and lacks the variableregion. Methods for preparing such fusions are disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted asmultimeric molecules wherein the F_(C) portions are disulfide bonded toeach other and two non-Ig polypeptides are arrayed in closed proximityto each other. Fusions of this type can be used for example, fordimerization, increasing stability and in vivo half-life, to affinitypurify ligand, as in vitro assay tool or antagonist. For use in assays,the chimeras are bound to a support via the F_(C) region and used in anELISA format.

A zalpha11 Ligand-binding polypeptide can also be used for purificationof ligand. The polypeptide is immobilized on a solid support, such asbeads of agarose, cross-linked agarose, glass, cellulosic resins,silica-based resins, polystyrene, cross-linked polyacrylamide, or likematerials that are stable under the conditions of use. Methods forlinking polypeptides to solid supports are known in the art, and includeamine chemistry, cyanogen bromide activation, N-hydroxysuccinimideactivation, epoxide activation, sulfhydryl activation, and hydrazideactivation. The resulting medium will generally be configured in theform of a column, and fluids containing ligand are passed through thecolumn one or more times to allow ligand to bind to the receptorpolypeptide. The ligand is then eluted using changes in saltconcentration, chaotropic agents (guanidine HCl), or pH to disruptligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40,1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.Alternatively, ligand/receptor binding can be analyzed using SELDI(™)technology (Ciphergen, Inc., Palo Alto, Calif.).

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

Zalpha11 Ligand polypeptides can also be used to prepare antibodies thatbind to zalpha11 Ligand epitopes, peptides or polypeptides. The zalpha11Ligand polypeptide or a fragment thereof serves as an antigen(immunogen) to inoculate an animal and elicit an immune response. One ofskill in the art would recognize that antigenic, epitope-bearingpolypeptides contain a sequence of at least 6, preferably at least 9,and more preferably at least 15 to about 30 contiguous amino acidresidues of a zalpha11 Ligand polypeptide (e.g., SEQ ID NO:2).Polypeptides comprising a larger portion of a zalpha11 Ligandpolypeptide, i.e., from 30 to 100 residues up to the entire length ofthe amino acid sequence are included. Antigens or immunogenic epitopescan also include attached tags, adjuvants and carriers, as describedherein. Suitable antigens include the zalpha11 Ligand polypeptideencoded by SEQ ID NO:2 from amino acid number 32 to amino acid number162, or a contiguous 9 to 131 amino acid fragment thereof. Othersuitable antigens include, the full length and the mature zalpha11Ligand, helices A-D, and individual or multiple helices A, B, C, and D,of the zalpha11 Ligand four-helical-bundle structure, as describedherein. Preferred peptides to use as antigens are hydrophilic peptidessuch as those predicted by one of skill in the art from a hydrophobicityplot, as described herein, for example, amino acid residues 114-119,101-105, 126-131, 113-118, and 158-162 of SEQ ID NO: 2.

Antibodies from an immune response generated by inoculation of an animalwith these antigens can be isolated and purified as described herein.Methods for preparing and isolating polyclonal and monoclonal antibodiesare well known in the art. See, for example, Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; andHurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats with a zalpha11 Ligand polypeptide or a fragment thereof.The immunogenicity of a zalpha11 Ligand polypeptide may be increasedthrough the use of an adjuvant, such as alum (aluminum hydroxide) orFreund's complete or incomplete adjuvant. Polypeptides useful forimmunization also include fusion polypeptides, such as fusions ofzalpha11 Ligand or a portion thereof with an immunoglobulin polypeptideor with maltose binding protein. The polypeptide immunogen may be afull-length molecule or a portion thereof. If the polypeptide portion is“hapten-like”, such portion may be advantageously joined or linked to amacromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication WO98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

Antibodies are considered to be specifically binding if: 1) they exhibita threshold level of binding activity, and 2) they do not significantlycross-react with related polypeptide molecules. A threshold level ofbinding is determined if anti-zalpha11 Ligand antibodies herein bind toa zalpha11 Ligand polypeptide, peptide or epitope with an affinity atleast 10-fold greater than the binding affinity to control (non-zalpha11Ligand) polypeptide. It is preferred that the antibodies exhibit abinding affinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10₈ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672,1949).

Whether anti-zalpha11 Ligand antibodies do not significantly cross-reactwith related polypeptide molecules is shown, for example, by theantibody detecting zalpha11 Ligand polypeptide but not known relatedpolypeptides using a standard Western blot analysis (Ausubel et al.,ibid.). Examples of known related polypeptides are those disclosed inthe prior art, such as known orthologs, and paralogs, and similar knownmembers of a protein family. Screening can also be done using non-humanzalpha11 Ligand, and zalpha11 Ligand mutant polypeptides. Moreover,antibodies can be “screened against” known related polypeptides, toisolate a population that specifically binds to the zalpha11 Ligandpolypeptides. For example, antibodies raised to zalpha11 Ligand areadsorbed to related polypeptides adhered to insoluble matrix; antibodiesspecific to zalpha11 Ligand will flow through the matrix under theproper buffer conditions. Screening allows isolation of polyclonal andmonoclonal antibodies non-crossreactive to known closely relatedpolypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.),Cold Spring Harbor Laboratory Press, 1988; Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995). Screening and isolation of specificantibodies is well known in the art. See, Fundamental Immunology, Paul(eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98,1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W.(eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol.2: 67-101, 1984. Specifically binding anti-zalpha11 Ligand antibodiescan be detected by a number of methods in the art, and disclosed below.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which bind to zalpha11 Ligand proteins orpolypeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutantzalpha11 Ligand protein or polypeptide.

Antibodies to zalpha11 Ligand may be used for tagging cells that expresszalpha11 Ligand; for isolating zalpha11 Ligand by affinity purification;for diagnostic assays for determining circulating levels of zalpha11Ligand polypeptides; for detecting or quantitating soluble zalpha11Ligand as a marker of underlying pathology or disease; in analyticalmethods employing FACS; for screening expression libraries; forgenerating anti-idiotypic antibodies; and as neutralizing antibodies oras antagonists to block zalpha11 Ligand activity in vitro and in vivo.Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to zalpha11 Ligand or fragments thereof may be usedin vitro to detect denatured zalpha11 Ligand or fragments thereof inassays, for example, Western Blots or other assays known in the art.

Suitable detectable molecules may be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like. Suitable cytotoxic moleculesmay be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria, toxin,saporin, Pseudomonas exotoxin, ricin, abrin and the like), as well astherapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90(either directly attached to the polypeptide or antibody, or indirectlyattached through means of a chelating moiety, for instance).Polypeptides or antibodies may also be conjugated to cytotoxic drugs,such as adriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

Binding polypeptides can also act as zalpha11 Ligand “antagonists” toblock zalpha11 Ligand binding and signal transduction in vitro and invivo. These anti-zalpha11 Ligand binding polypeptides would be usefulfor inhibiting zalpha11 Ligand activity or protein-binding.

Polypeptide-toxin fusion proteins or antibody-toxin fusion proteins canbe used for targeted cell or tissue inhibition or ablation (forinstance, to treat cancer cells or tissues). Alternatively, if thepolypeptide has multiple functional domains (i.e., an activation domainor a receptor binding domain, plus a targeting domain), a fusion proteinincluding only the targeting domain may be suitable for directing adetectable molecule, a cytotoxic molecule or a complementary molecule toa cell or tissue type of interest. In instances where the domain onlyfusion protein includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

Zalpha11 Ligand cytokine fusion proteins or antibody-cytokine fusionproteins can be used for enhancing in vivo killing of target tissues(for example, blood and bone marrow cancers), if the zalpha11 Ligandpolypeptide or anti-zalpha11 Ligand antibody targets thehyperproliferative blood or bone marrow cell (See, generally, Hornick etal., Blood 89:4437-47, 1997). The described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of cytokine. Suitable zalpha11 Ligandpolypeptides or anti-zalpha11 Ligand antibodies target an undesirablecell or tissue (i.e., a tumor or a leukemia), and the fused cytokinemediated improved target cell lysis by effector cells. Suitablecytokines for this purpose include interleukin 2 andgranulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.

Differentiation is a progressive and dynamic process, beginning withpluripotent stem cells and ending with terminally differentiated cells.Pluripotent stem cells that can regenerate without commitment to alineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products, and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population.

There is evidence to suggest that factors that stimulate specific celltypes down a pathway towards terminal differentiation ordedifferentiation affect the entire cell population originating from acommon precursor or stem cell. Thus, the present invention includesstimulating or inhibiting the proliferation of lymphoid cells,hematopoietic cells and epithelial cells.

Zalpha11 Ligand was isolated from tissue known to have importantimmunological function and which contain cells that play a role in theimmune system. Zalpha11 Ligand is expressed in CD3+ selected, activatedperipheral blood cells, and it has been shown that zalpha11 Ligandexpression increases after T cell activation. Moreover, results ofexperiments described in the Examples section herein demonstrate thatpolypeptides of the present invention have an effect on thegrowth/expansion and/or differentiated state of NK cells or NKprogenitors. Additional evidence demonstrates that zalpha11 Ligandaffects proliferation and/or differentiation of T cells and B cells invivo. Factors that both stimulate proliferation of hematopoieticprogenitors and activate mature cells are generally known. NK cells areresponsive to IL-2 alone, but proliferation and activation generallyrequire additional growth factors. For example, it has been shown thatIL-7 and Steel Factor (c-kit ligand) were required for colony formationof NK progenitors. IL-15 +IL-2 in combination with IL-7 and Steel Factorwas more effective (Mrózek et al., Blood 87:2632-2640, 1996). However,unidentified cytokines may be necessary for proliferation of specificsubsets of NK cells and/or NK progenitors (Robertson et. al., Blood76:2451-2438, 1990). A composition comprising zalpha11 Ligand and IL-15stimulates NK progenitors and NK cells, with evidence that thiscomposition is more potent than previously described factors andcombinations of factors.

Assays measuring differentiation include, for example, measuring cellmarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference). Alternatively, zalpha11 Ligand polypeptide itselfcan serve as an additional cell-surface or secreted marker associatedwith stage-specific expression of a tissue. As such, direct measurementof zalpha11 Ligand polypeptide, or its loss of expression in a tissue asif differentiates, can serve as a marker for differentiation of tissues.

Similarly, direct measurement of zalpha11 Ligand polypeptide, or itsloss of expression in a tissue can be determined in a tissue or in cellsas they undergo tumor progression. Increases in invasiveness andmotility of cells, or the gain or loss of expression of zalpha11 Ligandin a pre-cancerous or cancerous condition, in comparison to normaltissue, can serve as a diagnostic for transformation, invasion andmetastasis in tumor progression. As such, knowledge of a tumor's stageof progression or metastasis will aid the physician in choosing the mostproper therapy, or aggressiveness of treatment, for a given individualcancer patient. Methods of measuring gain and loss of expression (ofeither mRNA or protein) are well known in the art and described hereinand can be applied to zalpha11 Ligand expression. For example,appearance or disappearance of polypeptides that regulate cell motilitycan be used to aid diagnosis and prognosis of prostate cancer (Banyard,J. and Zetter, B. R., Cancer and Metast. Rev. 17:449-458, 1999). As aneffector of cell motility, zalpha11 Ligand gain or loss of expressionmay serve as a diagnostic for lymphoid, B-cell, epithelial,hematopoietic and other cancers.

Moreover, the activity and effect of zalpha11 Ligand on tumorprogression and metastasis can be measured in vivo. Several syngeneicmouse models have been developed to study the influence of polypeptides,compounds or other treatments on tumor progression. In these models,tumor cells passaged in culture are implanted into mice of the samestrain as the tumor donor. The cells will develop into tumors havingsimilar characteristics in the recipient mice, and metastasis will alsooccur in some of the models. Appropriate tumor models for our studiesinclude the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma(ATCC No. CRL-6323), amongst others. These are both commonly used tumorlines, syngeneic to the C57BL6/J mouse, that are readily cultured andmanipulated in vitro. Tumors resulting from implantation of either ofthese cell lines are capable of metastasis to the lung in C57BL6/J mice.The Lewis lung carcinoma model has recently been used in mice toidentify an inhibitor of angiogenesis (O'Reilly M S, et al. Cell 79:315-328,1994). C57BL6/J mice are treated with an experimental agenteither through daily injection of recombinant protein, agonist orantagonist or a one time injection of recombinant adenovirus. Three daysfollowing this treatment, 105 to 106 cells are implanted under thedorsal skin. Alternatively, the cells themselves may be infected withrecombinant adenovirus, such as one expressing zalpha11 Ligand, beforeimplantation so that the protein is synthesized at the tumor site orintracellularly, rather than systemically. The mice normally developvisible tumors within 5 days. The tumors are allowed to grow for aperiod of up to 3 weeks, during which time they may reach a size of1500-1800 mm³ in the control treated group. Tumor size and body weightare carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., zalpha11Ligand, on the ability of the tumor to recruit vasculature and undergometastasis can thus be assessed. In addition, aside from usingadenovirus, the implanted cells can be transiently transfected withzalpha11 Ligand. Use of stable zalpha11 Ligand transfectants as well asuse of induceable promoters to activate zalpha11 Ligand expression invivo are known in the art and can be used in this system to assesszalpha11 Ligand induction of metastasis. Moreover, purified zalpha11Ligand or zalpha11 Ligand conditioned media can be directly injected into this mouse model, and hence be used in this system. For generalreference see, O'Reilly M S, et al. Cell 79:315-328, 1994; and RuscianoD, et al. Murine Models of Liver Metastasis. Invasion Metastasis14:349-361, 1995.

Zalpha11 Ligand will be useful in treating tumorgenesis, and thereforewould be useful in the treatment of cancer. Zalpha11 Ligand inhibitsIL-4 stimulated proliferation of anti-IgM stimulated normal B-cells anda similar effect is observed in B-cell tumor lines suggesting that theremay be therapeutic benefit in treating patients with the zalpha11 Ligandin order to induce the B cell tumor cells into a less proliferativestate. The ligand could be administered in combination with other agentsalready in use including both conventional chemotherapeutic agents aswell as immune modulators such as interferon alpha. Alpha/betainterferons have been shown to be effective in treating some leukemiasand animal disease models, and the growth inhibitory effects ofinterferon-alpha and zalpha11 Ligand are additive for at least oneB-cell tumor-derived cell line.

The present invention provides a method of reducing proliferation of aneoplastic B or T cells comprising administering to a mammal with a B orT cell neoplasm an amount of a composition of zalpha11 Ligand sufficientto reduce proliferation of the neoplastic B or T cells. In otherembodiments, the composition can comprise at least one other cytokineselected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3ligand or stem cell factor.

In another aspect, the present invention provides a method of reducingproliferation of a neoplastic B or T cells comprising administering to amammal with a B or T cell neoplasm an amount of a composition ofzalpha11 Ligand antagonist sufficient to reducing proliferation of theneoplastic B or T cells. In other embodiments, the composition cancomprise at least one other cytokine selected from the group consistingof IL-2, IL-15, IL-4, GM-CSF, Flt3 ligand or stem cell factor.Furthermore, the zalpha11 Ligand antagonist can be a ligand/toxin fusionprotein.

A zalpha11 Ligand-saporin fusion toxin may be employed against a similarset of leukemias and lymphomas, extending the range of leukemias thatcan be treated with zalpha11 Ligand. Fusion toxin mediated activation ofthe zalpha11 receptor provides two independent means to inhibit thegrowth of the target cells, the first being identical to the effectsseen by the ligand alone, and the second due to delivery of the toxinthrough receptor internalization. The lymphoid restricted expressionpattern of the zalpha11 receptor suggests that the ligand-saporinconjugate can be tolerated by patients.

When treatment for malignancies includes allogeneic bone marrow or stemcell transplantion, zalpha11 Ligand may be valuable in enhancement ofthe graft-vs-tumor effect. zalpha11 Ligand stimulates the generation oflytic NK cells from marrow progenitors and stimulates the proliferationof T-cells following activation of the antigen receptors. Therefore,when patients receive allogenic marrow transplants, zalpha11 Ligand willenhance the generation of anti-tumor responses, with or without theinfusion of donor lymphocytes.

The tissue distribution of a receptor for a given cytokine offers astrong indication of the potential sites of action of that cytokine.Northern analysis of zalpha11 receptor revealed transcripts in humanspleen, thymus, lymph node, bone marrow, and peripheral bloodleukocytes. Specific cell types were identified as expressing zalpha11receptors, and strong signals were seen in a mixed lymphocyte reaction(MLR) and in the Burkitt's lymphoma Raji. The two monocytic cell lines,THP-1 (Tsuchiya et al., Int. J. Cancer 26:171-176, 1980) and U937(Sundstrom et al., Int. J. Cancer 17:565-577, 1976), were negative.

Zalpha11 receptor is expressed at relatively high levels in the MLR, inwhich peripheral blood mononuclear cells (PBMNC) from two individualsare mixed, resulting in mutual activation. Detection of high levels oftranscript in the MLR but not in resting T or B cell populationssuggests that zalpha11 receptor expression may be induced in one or morecell types during activation. Activation of isolated populations of Tand B cells can be artificially achieved by stimulating cells with PMAand ionomycin. When sorted cells were subjected to these activationconditions, levels of zalpha11 receptor transcript increased in bothcell types, supporting a role for this receptor and zalpha11 Ligand inimmune responses, especially in autocrine and paracrine T and B cellexpansions during activation. Zalpha11 Ligand may also play a role inthe expansion of more primitive progenitors involved in lymphopoiesis.

zalpha11 receptor was found to be present at low levels in resting T andB cells, and was upregulated during activation in both cell types.Interestingly, the B cells also down-regulate the message more quicklythan do T cells, suggesting that amplitude of signal and timing ofquenching of signal are important for the appropriate regulation of Bcell responses.

In addition, a large proportion of intestinal lamina propria cells showpositive hybridization signals with zalpha11 receptor. This tissueconsists of a mixed population of lymphoid cells, including activatedCD4+ T cells and activated B cells. Immune dysfunction, in particularchronic activation of the mucosal immune response, plays an importantrole in the etiology of Crohn's disease; abnormal response to and/orproduction of proinflammatory cytokines is also a suspected factor(Braegger et al., Annals Allergy 72:135-141, 1994; Sartor R B Am. J.Gastroenterol. 92:5S-11 S, 1997. zalpha11 Ligand in concert with IL-15expands NK cells from bone marrow progenitors and augments NK celleffector function. zalpha11 Ligand also co-stimulates mature B cellsstimulated with anti-CD40 antibodies, but inhibits B cell proliferationto signals through IgM. zalpha11 Ligand enhances T cell proliferation inconcert with a signal through the T cell receptor, and overexpression intransgenic mice leads to lymphopenia and an expansion of monocytes andgranulocytes. These pleiotropic effects of zalpha11 Ligand suggest thatit can provide therapeutic utility for a wide range of diseases arisingfrom defects in the immune system, including (but not limited to)systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiplesclerosis (MS), myasthenia gravis, and diabetes. It is important to notethat these diseases are the result of a complex network of immunedysfunction (SLE, for example, is the manifestation of defects in both Tand B cells), and that immune cells are dependent upon interaction withone another to elicit a potent immune response. Therefore, zalpha11Ligand (or an antagonist of the Ligand) that can be used to manipulatemore than one type of immune cell is an attractive therapeutic candidatefor intervention at multiple stages of disease.

The polypeptides and proteins of the present invention can also be usedex vivo, such as in autologous marrow culture. Briefly, bone marrow isremoved from a patient prior to chemotherapy or organ transplant andtreated with zalpha11 Ligand, optionally in combination with one or moreother cytokines. The treated marrow is then returned to the patientafter chemotherapy to speed the recovery of the marrow or aftertransplant to suppress graft vs. Host disease. In addition, the proteinsof the present invention can also be used for the ex vivo expansion ofmarrow or peripheral blood progenitor (PBPC) cells. Prior to treatment,marrow can be stimulated with stem cell factor (SCF) to release earlyprogenitor cells into peripheral circulation. These progenitors can becollected and concentrated from peripheral blood and then treated inculture with zalpha11 Ligand, optionally in combination with one or moreother cytokines, including but not limited to SCF, IL-2, IL-4, IL-7 orIL-15, to differentiate and proliferate into high-density lymphoidcultures, which can then be returned to the patient followingchemotherapy or transplantation.

The present invention provides a method for expansion of hematopoieticcells and hematopoietic cell progenitors comprising culturing bonemarrow or peripheral blood cells with a composition comprising an amountof zalpha11 Ligand sufficient to produce an increase in the number oflymphoid cells in the bone marrow or peripheral blood cells as comparedto bone marrow or peripheral blood cells cultured in the absence ofzalpha11 Ligand. In other embodiments., the hematopoietic cells andhematopoietic progenitor cells are lymphoid cells. In anotherembodiment, the lymphoid cells are NK cells or cytotoxic T cells.Furthermore, the composition can also comprise at least one othercytokine selected from the group consisting of IL-2, IL-15, IL-4,GM-CSF, Flt3 ligand and stem cell factor.

Alternatively, zalpha11 Ligand may activate the immune system whichwould be important in boosting immunity to infectious diseases, treatingimmunocompromised patients, such as HIV+ patients, or in improvingvaccines. In particular, zalpha11 Ligand stimulation or expansion of NKcells, or their progenitors, would provide therapeutic value intreatment of viral infection, and as an anti-neoplastic factor. NK cellsare thought to play a major role in elimination of metastatic tumorcells and patients with both metastases and solid tumors have decreasedlevels of NK cell activity (Whiteside et. al., Curr. Top. Microbiol.Immunol. 230:221-244, 1998). Similarly, zalpha11 Ligand stimulation ofthe immune response against viral and non-viral pathogenic agents(including bacteria, protozoa, and fungi) would provide therapeuticvalue in treatment of such infections by inhibiting the growth of suchinfections agents. Determining directly or indirectly the levels of apathogen or antigen, such as a tumor cell, present in the body can beachieved by a number of methods known in the art and described herein.

The present invention include a method of stimulating an immune responsein a mammal exposed to an antigen or pathogen comprising the steps of:(1) determining directly or indirectly the level of antigen or pathogenpresent in said mammal; (2) administering a composition comprisingzalpha11 Ligand polypeptide in an acceptable pharmaceutical vehicle; (3)determining directly or indirectly the level of antigen or pathogen insaid mammal; and (4) comparing the level of the antigen or pathogen instep 1 to the antigen or pathogen level in step 3, wherein a change inthe level is indicative of stimulating an immune response. In anotherembodiment the zalpha11 Ligand composition is re-administered. In otherembodiments, the antigen is a B cell tumor; a virus; a parasite or abacterium.

In another aspect, the present invention provides a method ofstimulating an immune response in a mammal exposed to an antigen orpathogen comprising: (1) determining a level of an antigen- orpathogen-specific antibody; (2) administering a composition comprisingzalpha11 Ligand polypeptide in an acceptable pharmaceutical vehicle; (3)determining a post administration level of antigen- or pathogen-specificantibody; (4) comparing the level of antibody in step (1) to the levelof antibody in step (3), wherein an increase in antibody level isindicative of stimulating an immune response.

Polynucleotides encoding zalpha11 Ligand polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzalpha11 Ligand activity. If a mammal has a mutated or absent zalpha11Ligand gene, the zalpha11 Ligand gene can be introduced into the cellsof the mammal. In one embodiment, a gene encoding a zalpha11 Ligandpolypeptide is introduced in vivo in a viral vector. Such vectorsinclude an attenuated or defective DNA virus, such as, but not limitedto, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus(EBV), adenovirus, adeno-associated virus (AAV), and the like. Defectiveviruses, which entirely or almost entirely lack viral genes, arepreferred. A defective virus is not infective after introduction into acell. Use of defective viral vectors allows for administration to cellsin a specific, localized area, without concern that the vector caninfect other cells. Examples of particular vectors include, but are notlimited to, a defective herpes simplex virus 1 (HSVI) vector (Kaplitt etal., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirusvector, such as the vector described by Stratford-Perricaudet et al., J.Clin. Invest. 90:626-30, 1992; and a defective adeno-associated virusvector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et al.,J. Virol. 63:3822-8, 1989).

A zalpha11 Ligand gene can be introduced in a retroviral vector, e.g.,as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann et al.Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al.,U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988;Temin et al., U.S. Pat. No. 5,124,263; International Patent PublicationNo. WO 95/07358, published Mar. 16, 1995 by Dougherty et al.; and Kuo etal., Blood 82:845, 1993. Alternatively, the vector can be introduced bylipofection in vivo using liposomes. Synthetic cationic lipids can beused to prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987;Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). The use oflipofection to introduce exogenous genes into specific organs in vivohas certain practical advantages. Molecular targeting of liposomes tospecific cells represents one area of benefit. More particularly,directing transfection to particular cells represents one area ofbenefit. For instance, directing transfection to particular cell typeswould be particularly advantageous in a tissue with cellularheterogeneity, such as the immune system, pancreas, liver, kidney, andbrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Antisense methodology can be used to inhibit zalpha11 Ligand genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a zalpha11Ligand-encoding polynucleotide (e.g., a polynucleotide as set froth inSEQ ID NO:1) are designed to bind to zalpha11 Ligand-encoding mRNA andto inhibit translation of such mRNA. Such antisense polynucleotides areused to inhibit expression of zalpha11 Ligand polypeptide-encoding genesin cell culture or in a subject.

Mice engineered to express the zalpha11 Ligand gene, referred to as“transgenic mice,” and mice that exhibit a complete absence of zalpha11Ligand gene function, referred to as “knockout mice,” may also begenerated (Snouwaert et al., Science 257:1083, 1992; Lowell et al.,Nature 366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express zalpha11 Ligand, either ubiquitouslyor under a tissue-specific or tissue-restricted promoter can be used toask whether over-expression causes a phenotype. For example,over-expression of a wild-type zalpha11 Ligand polypeptide, polypeptidefragment or a mutant thereof may alter normal cellular processes,resulting in a phenotype that identifies a tissue in which zalpha11Ligand expression is functionally relevant and may indicate atherapeutic target for the zalpha11 Ligand, its agonists or antagonists.For example, a preferred transgenic mouse to engineer is one thatover-expresses the zalpha11 Ligand (amino acid residues 32-162 of SEQ IDNO:2). Moreover, such over-expression may result in a phenotype thatshows similarity with human diseases. Similarly, knockout zalpha11Ligand mice can be used to determine where zalpha11 Ligand is absolutelyrequired in vivo. The phenotype of knockout mice is predictive of the invivo effects of that a zalpha11 Ligand antagonist, such as thosedescribed herein, may have. The human or mouse zalpha11 Ligand cDNA canbe used to generate knockout mice. These mice may be employed to studythe zalpha11 Ligand gene and the protein encoded thereby in an in vivosystem, and can be used as in vivo models for corresponding humandiseases. Moreover, transgenic mice expression of zalpha11 Ligandantisense polynucleotides or ribozymes directed against zalpha11 Ligand,described herein, can be used analogously to transgenic mice describedabove. Studies may be carried out by administration of purified zalpha11Ligand protein, as well.

For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. The bioactive polypeptide orantibody conjugates described herein can be delivered intravenously,intraarterially or intraductally, or may be introduced locally at theintended site of action. Intravenous administration will be by bolusinjection or infusion over a typical period of one to several hours. Ingeneral, pharmaceutical formulations will include a zalpha11 Ligandprotein in combination with a pharmaceutically acceptable vehicle, suchas saline, buffered saline, 5% dextrose in water or the like.Formulations may further include one or more excipients, preservatives,solubilizers, buffering agents, albumin to prevent protein loss on vialsurfaces, etc. Methods of formulation are well known in the art and aredisclosed, for example, in Remington: The Science and Practice ofPharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa., 19th ed.,1995. Therapeutic doses will generally be in the range of 0.1 to 100μg/kg of patient weight per day, preferably 0.5-20 μg/kg per day, withthe exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. The proteins may be administered foracute treatment, over one week or less, often over a period of one tothree days or may be used in chronic treatment, over several months oryears. In general, a therapeutically effective amount of zalpha11 Ligandis an amount sufficient to produce a clinically significant change inhematopoietic or immune function.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Construction of MPL-Zalpha11 Polypeptide Chimera: MPLExtracellular and TM Domain Fused to the Zalpha11 IntracellularSignaling Domain

The extracellular and transmembrane domains of the murine MPL receptorwere isolated from a plasmid containing the murine MPL receptor(PHZ1/MPL plasmid) using PCR with primers ZC17,212 (SEQ ID NO:5) andZC19,914 (SEQ ID NO:6). The reaction conditions were as follows: 95° C.for 1 min.; 35 cycles at 95° C. for 1 min., 45° C. for 1 min., 72° C.for 2 min.; followed by 72° C. at 10 min.; then a 10° C. soak. The PCRproduct was run on a 1% low melting point agarose (Boerhinger Mannheim,Indianapolis, Ind.) and the approximately 1.5 kb MPL receptor fragmentisolated using Qiaquick™ gel extraction kit (Qiagen) as permanufacturer's instructions.

The intracellular domains of human zalpha11 were isolated from a plasmidcontaining zalpha11 receptor cDNA using PCR with primers ZC19,913 (SEQID NO:8) and ZC20,097 (SEQ ID NO:9). The polynucleotide sequencecorresponding to the zalpha11 receptor coding sequence is shown in SEQID NO:7, and the corresponding amino acid sequence shown in SEQ IDNO:115. The reaction conditions were as per above. The PCR product wasrun on a 1% low melting point agarose (Boerhinger Mannheim) and theapproximately 900 bp zalpha11 fragment isolated using Qiaquick gelextraction kit as per manufacturer's instructions.

Each of the isolated fragments described above were mixed at a 1:1volumetric ratio and used in a PCR reaction using ZC17,212 (SEQ ID NO:5)and ZC20,097 (SEQ ID NO:9) to create the MPL-zalpha11 chimera. Thereaction conditions were as follows: 95° C. for 1 min.; 35 cycles at 95°C. for 1 min., 55° C. for 1 min., 72° C. for 2 min.; followed by 72° C.at 10 min.; then a 10° C. soak. The entire PCR product was run on a 1%low melting point agarose (Boehringer Mannheim) and the approximately2.4 kb MPL-zalpha11 chimera fragment isolated using Qiaquick gelextraction kit (Qiagen) as per manufacturer's instructions. TheMPL-zalpha11 chimera fragment was digested with EcoRI (BRL) and XbaI(Boerhinger Mannheim) as per manufacturer's instructions. The entiredigest was run on a 1% low melting point agarose (Boehringer Mannheim)and the cleaved MPL-zalpha11 chimera isolated using Qiaquick™ gelextraction kit (Qiagen) as per manufacturer's instructions. Theresultant cleaved MPL-zalpha11 chimera was inserted into an expressionvector as described below.

Recipient expression vector pZP-5N was digested with EcoRI (BRL) andHindIII (BRL) as per manufacturer's instructions, and gel purified asdescribed above. This vector fragment was combined with the EcoRI andXbaI cleaved MPL-zalpha11 chimera isolated above and a XbaI/HindIIIlinker fragment in a ligation reaction. The ligation was run using T4Ligase (BRL), at 15° C. overnight. A sample of the ligation waselectroporated in to DH10B ElectroMAX™ electrocompetent E. coli cells(25 μF, 200 Ω, 2.3V). Transformants were plated on LB+Ampicillin platesand single colonies screened by PCR to check for the MPL-zalpha11chimera using ZC17,212 (SEQ ID NO:5) and ZC20,097 (SEQ ID NO:9) usingthe PCR conditions as described above.

Confirmation of the MPL-zalpha11 chimera sequence was made by sequenceanalyses using the following primers: ZC12,700 (SEQ ID NO: 10), ZC5,020(SEQ ID NO:11), ZC6,675 (SEQ ID NO:12), ZC7,727 (SEQ ID NO:13), ZC8,290(SEQ ID NO:14), ZC19,572 (SEQ ID NO:15), ZC6,622 (SEQ ID NO:16), ZC7,736(SEQ ID NO:17), and ZC9,273 (SEQ ID NO:18). The insert was approximately2.4 bp, and was full-length.

Example 2 MPL-Zalpha11 Chimera Based Proliferation in BAF3 Assay UsingAlamar Blue

A. Construction of BaF3 Cells Expressing MPL-Zalpha11 Chimera

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2 ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM L-glutaMax-1™(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics(GIBCO BRL)). Prior to electroporation, pZP-5N/MPL-zalpha11 plasmid DNA(Example 1) was prepared and purified using a Qiagen Maxi Prep kit(Qiagen) as per manufacturer's instructions. BaF3 cells forelectroporation were washed once in RPMI media and then resuspended inRPMI media at a cell density of 10⁷ cells/ml. One ml of resuspended BaF3cells was mixed with 30 μg of the pZP-5N/MPL-zalpha11 plasmid DNA andtransferred to separate disposable electroporation chambers (GIBCO BRL).Following a 15 minute incubation at room temperature the cells weregiven two serial shocks (800 1Fad/300 V.; 1180 1Fad/300 V.) delivered byan electroporation apparatus (CELL-PORATOR™; GIBCO BRL). After a 5minute recovery time, the electroporated cells were transferred to 50 mlof complete media and placed in an incubator for 15-24 hours (37° C., 5%CO₂). The cells were then spun down and resuspended in 50 ml of completemedia containing Geneticin™ (Gibco) selection (500 μg/ml G418) in aT-162 flask to isolate the G418-resistant pool. Pools of the transfectedBaF3 cells, hereinafter called BaF3/MPL-zalpha11 cells, were assayed forsignaling capability as described below.

B. Testing the Signaling Capability of the BaF3/MPL-Zalpha11 Cells Usingan Alamar Blue Proliferation Assay

BaF3/MPL-zalpha11 cells were spun down and washed in the complete media,described above, but without mIL-3 (hereinafter referred to as “mIL-3free media”). The cells were spun and washed 3 times to ensure theremoval of the mIL-3. Cells were then counted in a hemacytometer. Cellswere plated in a 96-well format at 5000 cells per well in a volume of100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/MPL-zalpha11 cells was assessed using murinemthrombopoietin (mTPO) diluted with mIL-3 free media to 500 ng/ml,250ng/ml, 125 ng/ml, 62 ng/ml, 30 ng/ml, 15 ng/ml, 7.5 ng/ml, 3.75ng/ml, 1.8 ng/ml, 0.9 ng/ml, 0.5 ng/ml and 0.25 ng/ml concentrations.100 μl of the diluted mTPO was added to the BaF3/MPL-zalpha11 cells. Thetotal assay volume is 200 μl. Negative controls were run in parallelusing mIL-3 free media only, without the addition of mTPO. The assayplates were incubated at 37° C., 5% CO₂ for 3 days at which time AlamarBlue (Accumed, Chicago, Ill.) was added at 20 μl/well. Alamar Blue givesa fluourometric readout based on the metabolic activity of cells, and isthus a direct measurement of cell proliferation in comparison to anegative control. Plates were again incubated at 37° C., 5% CO₂ for 24hours. Plates were read on the Fmax™ plate reader (Molecular DevicesSunnyvale, Calif.) using the SoftMaX™ Pro program, at wavelengths 544(Excitation) and 590 (Emmission).

Results confirmed the signaling capability of the intracellular portionof the zalpha11 receptor, as the thrombopoietin induced proliferation atapproximately 10 fold over back ground at mTPO concentrations of 62ng/ml and greater.

Example 3 Construction of Expression Vector Expressing Full-LengthZalpha11

The entire zalpha11 receptor was isolated from a plasmid containingzalpha11 receptor cDNA (SEQ ID NO:7) using PCR with primers ZC19,905(SEQ ID NO:19) and ZC19,906 (SEQ ID NO:20). The reaction conditions wereas follows: 95° C. for min; 35 cycles at 95° C. for 1 min, 55° C. for 1min, 72° C. for 2 min; followed by 72° C. at 10 min; then a 10° C. soak.The PCR product was run on a 1% low melting point agarose (BoerhingerMannheim) gel and the approximately 1.5 kb zalpha11 cDNA isolated usingQiaquick™ gel extraction kit (Qiagen) as per manufacturer'sinstructions.

The purified zalpha11 cDNA was digested with BamHI (Boerhinger Mannheim)and EcoRI (BRL) as per manufacturer's instructions. The entire digestwas run on a 1% low melting point agarose (Boerhinger Mannheim) gel andthe cleaved zalpha11 fragment was purified the using Qiaquick™ gelextraction kit as per manufacturer's instructions. The resultant cleavedzalpha11 fragment was inserted into an expression vector as describedbelow.

Recipient expression vector pZP-5N was digested with BamHI (BoerhingerMannheim) and EcoRI (BRL) as per manufacturer's instructions, and gelpurified as described above. This vector fragment was combined with theBamHI and EcoRI cleaved zalpha11 fragment isolated above in a ligationreaction using T4 Ligase (BRL). The ligation was incubated at 15° C.overnight. A sample of the ligation was electroporated in to DH10BelectroMAX™ electrocompetent E. coli cells (25 μF, 200 Ω, 2.3V).Transformants were plated on LB+Ampicillin plates and single coloniesscreened by PCR to check for the zalpha11 sequence using ZC19,905 (SEQID NO: 19) and ZC19,906 (SEQ ID NO:20) using the PCR conditions asdescribed above.

Confirmation of the zalpha11 sequence was made by sequence analysesusing the following primers: ZC12,700 (SEQ ID NO:10), ZC5,020 (SEQ IDNO:11), ZC20,114 (SEQ ID NO:21), ZC19,459 (SEQ ID NO:22), ZC19,954 (SEQID NO:23), and ZC20,116 (SEQ ID NO:24). The insert was approximately 1.6kb, and was full-length.

Example 4 Zalpha11 Based Proliferation in BAF3 Assay Using Alamar Blue

A. Construction of BaF3 Cells Expressing Zalpha11 Receptor

BaF3 cells expressing the full-length zalpha11 receptor was constructedas per Example 2A above, using 30 μg of the zalpha11 expression vector,described in Example 3 above. The BaF3 cells expressing the zalpha11receptor mRNA were designated as BaF3/zalpha11. These cells were used toscreen for zalpha11 Ligand as described below in Examples 5 and 6.

Example 5 Screening for Zalpha11 Ligand Using BaF3/Zalpha11 Cells Usingan Alamar Blue Proliferation Assay

A. Activation of Primary Monkey Splenocytes to Test for Presence ofZalpha11 Ligand

Monkey splenocytes were stimulated in vitro to produce conditioned mediato test for the presence of zalpha11 Ligand activity as described below.Monkey spleens were obtained from 8 year old female M. nesestrianmonkeys. The spleens were teased part to produce a single cellsuspension. The mononuclear cells were isolated by Ficoll-Paque® PLUS(Pharmacia Biotech, Uppsala, Sweden) density gradient. The mononuclearcells were seeded at 2×10⁶ cells/ml in RPMI-1640 media supplemented with10% FBS and activated with 5 ng/ml Phorbol-12-myristate-13-acetate (PMA)(Calbiochem, San Diego, Calif.), and 0.5 mg/ml Ionomycin™ (Calbiochem)for 48 hrs. The supernatant from the stimulated monkey spleen cells wasused to assay proliferation of the BaF3/zalpha11 cells as describedbelow.

B. Screening for Zalpha11 Ligand using BaF3/Zalpha11 Cells using anAlamar Blue Proliferation Assay

BaF3/Zalpha11 cells were spun down and washed in mIL-3 free media. Thecells were spun and washed 3 times to ensure the removal of the mIL-3.Cells were then counted in a hemacytometer. Cells were plated in a96-well format at 5000 cells per well in a volume of 100 μl per wellusing the mIL-3 free media.

Proliferation of the BaF3/Zalpha11 cells was assessed using conditionedmedia from activated monkey spleen (see Example 5A). Conditioned mediawas diluted with mIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%,1.5%, 0.75% and 0.375% concentrations. 100 μl of the diluted conditionedmedia was added to the BaF3/Zalpha11 cells. The total assay volume is200 μl. The assay plates were incubated at 37° C., 5% CO₂ for 3 days atwhich time Alamar Blue (Accumed, Chicago, Ill.) was added at 20 μl/well.Plates were again incubated at 37° C., 5% CO₂ for 24 hours. Plates wereread on the Fmax™ plate reader (Molecular devices) as described above(Example 2).

Results confirmed the proliferative response of the BaF3/Zalpha11 cellsto a factor present in the activated monkey spleen conditioned media.The response, as measured, was approximately 4-fold over background atthe 50% concentration. The untransfected BaF3 cells did not proliferatein response to this factor, showing that this factor is specific for theZalpha11 receptor.

C. Human Primary Source Used to Isolate Zalpha11 Ligand

100 ml blood draws were taken from each of six donors. The blood wasdrawn using 10×10 ml vacutainer tubes containing heparin. Blood waspooled from six donors (600 ml), diluted 1:1 in PBS, and separated usinga Ficoll-Paque® PLUS (Pharmacia Biotech). The isolated primary humancell yield after separation on the ficoll gradient was 1.2×10⁹ cells.

Cells were suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA, 2 mM EDTA).1.6 ml of cell suspension was removed and 0.4 ml CD3 microbeads(Miltenyi Biotec, Auburn, Calif.) added. The mixture was incubated for15 min. at 4° C. These cells labeled with CD3 beads were washed with 30ml MACS buffer, and then resuspended in 2 ml MACS buffer.

A VS+ column (Miltenyi) was prepared according to the manufacturer'sinstructions. The VS+ column was then placed in a VarioMACS™ magneticfield (Miltenyi). The column was equilibrated with 5 ml MACS buffer. Theisolated primary human cells were then applied to the column. The CD3negative cells were allowed to pass through. The column was rinsed with9 ml (3×3 ml) MACS buffer. The column was then removed from the magnetand placed over a 15 ml falcon tube. CD3+ cells were eluted by adding 5ml MACS buffer to the column and bound cells flushed out using theplunger provided by the manufacturer. The incubation of the cells withthe CD3 magnetic beads, washes, and VS+ column steps (incubation throughelution) above were repeated five more times. The resulting CD3+fractions from the six column separations were pooled. The yield of CD3+selected human cells were 3×10⁸ total cells.

A sample of the pooled CD3+ selected human cells was removed forstaining and sorting on a fluorescent antibody cell sorter (FACS) toassess their purity. The human CD3+ selected cells were 91% CD3+ cells.

The human CD3+ selected cells were activated by incubating in RPMI+5%FBS+PMA 10 ng/ml and Ionomycin 0.5 μg/ml (Calbiochem) for 13 hours 37°C. The supernatant from these activated CD3+ selected human cells wastested for zalpha11 Ligand activity as described below. Moreover, theactivated CD3+ selected human cells were used to prepare a cDNA library,as described in Example 6, below.

D. Testing Supernatant From Activated CD3+ Selected Human Cells forZalpha11 Ligand Using BaF3/Zalpha11 Cells and an Alamar BlueProliferation Assay

BaF3/Zalpha11 cells were spun down and washed in mIL-3 free media. Thecells were spun and washed 3 times to ensure the removal of the mIL-3.Cells were then counted in a hemacytometer. Cells were plated in a96-well format at 5000 cells per well in a volume of 100 μl per wellusing the mIL-3 free media.

Proliferation of the BaF3/Zalpha11 cells was assessed using conditionedmedia from activated CD3+ selected human cells (see Example 5C) dilutedwith mIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and0.375% concentrations. 100 μl of the diluted conditioned media was addedto the BaF3/Zalpha11 cells. The total assay volume is 200 μl. The assayplates were incubated and assayed as described in Example 5B.

Results confirmed the proliferative response of the BaF3/Zalpha11 cellsto a factor present in the activated CD3+ selected human Cellconditioned media. The response, as measured, was approximately 10-foldover background at the 50% concentration. The untransfected BaF3 cellsdid not proliferate in response to this factor, showing that this factoris specific for the Zalpha11 receptor. Moreover soluble alpha11 receptorblocked this proliferative activity in the BaF3/Zalpha11 cells (see,Example 11).

Example 6 Cloning of Human Zalpha11 Ligand from a Human CD3+ SelectedCell Library

Screening of a primary human activated CD3+ selected cell cDNA libraryrevealed an isolated cDNA that is a novel member of the four-helixbundle cytokine family. This cDNA encoded the zalpha11 Ligand. The cDNAwas identified by screening for activity of the zalpha11 Ligand usingthe zalpha11 receptor.

A. The Vector for CD3+ Selected Library Construction

The vector for CD3+ selected library construction was pZP7NX. The pZP7NXvector was constructed as follows: The coding region for the DHFRselective marker in vector pZP7 was removed by DNA digestion with NcoIand PstI restriction enzymes (Boehringer Mannheim). The digested DNA wasrun on 1% agarose gel, cut out and gel purified using the Qiagen GelExtraction Kit (Qiagen) as per manufacturer's instructions. A DNAfragment representing the coding region of Zeocin selective marker wasamplified by PCR method with primers ZC13,946 (SEQ ID NO:25) andZC13,945 (SEQ ID NO:26), and pZeoSV2(+) as a template. There areadditional PstI and BclI restriction sites in primer ZC13,946 (SEQ IDNO:25), and additional NcoI and SfuI sites in primer ZC13,945 (SEQ IDNO:26). The PCR fragment was cut with PstI and NcoI restriction enzymesand cloned into pZP7 vector prepared by cleaving with the same twoenzymes and subsequent gel purification. This vector was named pZP7Z.Then the Zeocin coding region was removed by DNA digestion of vectorpZP7Z with BclI and SfuI restriction enzymes. The digested DNA was runon 1% agarose gel, cut out and gel purified, and then ligated with a DNAfragment of Neomycin coding region cut from pZem228 vector (deposited atthe American Type Culture Collection (ATCC), Manassas, Va.; ATCC DepositNo. 69446) with the same restriction enzymes (BclI and SfuI).

This new vector was named pZP7N, in which the coding region for DHFRselective marker was replaced by the coding region for a Neomycinselective marker from vector pZem228. A stuffer fragment including anXhol site was added to pZP7N to create a vector suitable for highefficiency directional cloning of cDNA; this new vector was calledpZP7NX. To prepare the vector for cDNA, 20 μg of pZP7NX was digestedwith 20 units of EcoR1 (Life Technologies Gaithersberg, Md.) and 20units of Xhol (Boehringer Mannheim Indianapolis, Ind.) for 5 hours at37° C., then 68° C. for 15 minutes. The digest was then run on a 0.8%low melt agarose 1XTAE gel to separate the stuffer from the vector. Thevector band was excised and digested with “beta-Agarase” (New EnglandBiolabs, Beverly, Mass.) following the manufacturer's recommendations.After ethanol precipitation the digested vector was resuspended in waterto 45 ng/ml in preparation for ligation of CD3+ selected cDNA librarydescribed below.

B. Preparation of Primary Human Activated CD3+ Selected Cell cDNALibrary

Approximately 1.5×10⁸ primary human CD3+ selected cells stimulated inionomycin/PMA were isolated by centrifugation after culturing at 37° C.for 13 hours (Example 5C). Total RNA was isolated from the cell pelletusing the “RNeasy Midi” kit from Qiagen, Inc. (Valencia, Calif.). mRNAwas isolated from 225 micrograms of total RNA using the “MPG mRNApurification kit” from CPG Inc. (Lincoln Park, N.J.). 3.4 micrograms ofmRNA was isolated and converted to double stranded cDNA using thefollowing procedure.

First strand cDNA from stimulated human CD3+ selected cells wassynthesized as follows. Nine μl Oligo d(T)-selected poly(A) CD3+ RNA ata concentration of 0.34 μg/μl and 1.0 μl of 1 μg/μl first strand primerZC18,698 (SEQ ID NO:27) containing an XhoI restriction site were mixedand heated at 65° C. for 4 minutes and cooled by chilling on ice. Firststrand cDNA synthesis was initiated by the addition of 9 μl of firststrand buffer (5× SUPERSCRIPT® buffer; (Life Technologies), 4 μl of 100mM dithiothreitol and 2 μl of a deoxynucleotide triphosphate solutioncontaining 10 mM each of dATP, dGTP, dTTP and 5-methyl-dCTP (PharmaciaBiotech Inc.) to the RNA-primer mixture. The reaction mixture wasincubated at 45° C. for 4 minutes followed by the addition of 8 μl of200 U/μl SuperscriptII®, RNase H-reverse transcriptase (Lifetechnologies). The reaction was incubated at 45° C. for 45 minutesfollowed by an incubation ramp of 1° C. every 2 minutes to 50° C. wherethe reaction was held for 10 minutes. To denature any secondarystructure and allow for additional extension of the cDNA the reactionwas then heated to 70° C. for 2 minutes then dropped to 55° C. for 4minutes after which 2 μl of SuperscriptII® RT was added and incubated anadditional 15 minutes followed by a ramp up to 70° C. 1 minute/1° C.Unincorporated nucleotides were removed from the cDNA by twiceprecipitating in the presence of 2 μg of glycogen carrier, 2.0 Mammonium acetate and 2.5 volume ethanol, followed by a 100 μl wash with70% ethanol. The cDNA was resuspended in 98 μl water for use in secondstrand synthesis.

Second strand synthesis was performed on the first strand cDNA underconditions that promoted first strand priming of second strand synthesisresulting in DNA hairpin formation. The second strand reaction contained98 μl of the first strand cDNA, 30 μl of 5× polymerase I buffer (100 mMTris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl₂, 50 mM (NH4)2SO4), 2 μl of100 mM dithiothreitol, 6 μl of a solution containing 10 mM of eachdeoxynucleotide triphosphate, 5 μl of 5 mM b-NAD, 1 μl of 3 U/μl E. coliDNA ligase (New England Biolabs Inc.) and 4 μl of 10 U/μl E. coli DNApolymerase I (New England Biolabs Inc.). The reaction was assembled atroom temperature and was incubated at room temperature for 2 minutesfollowed by the addition of 4 μl of 3.8 U/μl RNase H (LifeTechnologies). The reaction was incubated at 15° C. for two hoursfollowed by a 15 minute incubation at room temperature. 10 μl of 1M TRISpH7.4 was added to the reaction and extracted twice withphenol/chloroform and once with chloroform, the organic phases were thenback extracted with 50 μl of TE (10 mM TRIS pH 7.4, 1 mM EDTA), pooledwith the other aqueous and ethanol precipitated in the presence of 0.3 Msodium acetate. The pellet was washed with 100 μl 70% ethanol air driedand resuspended in 40 μl water.

The single-stranded DNA of the hairpin structure was cleaved using mungbean nuclease. The reaction mixture contained 40 μl of second strandcDNA, 5 μl of 10× mung bean nuclease buffer (Life technologies), 5 μl ofmung bean nuclease (Pharmacia Biotech Corp.) diluted to 1 U/μl in 1×mung bean nuclease buffer. The reaction was incubated at 37° C. for 45minutes. The reaction was terminated by the addition of 10 ll of 1 MTris: HCl, pH 7.4 followed by sequential phenol/chloroform andchloroform extractions as described above. Following the extractions,the cDNA was ethanol precipitated in the presence of 0.3 M sodiumacetate. The pellet was washed with 100 μl 70% ethanol air dried andresuspended in 38 μl water.

The resuspended cDNA was blunt-ended with T4 DNA polymerase. The cDNA,which was resuspended in 38 μl of water, was mixed with 12 μl 5×T4 DNApolymerase buffer (250 mM Tris:HCl, pH 8.0, 250 mM KCl, 25 mM MgCl₂), 2μl 0.1 M dithiothreitol, 6 μl of a solution containing 10 mM of eachdeoxynucleotide triphosphate and 2 μl of 1 U/μl T4 DNA polymerase(Boehringer Mannheim Corp.). After an incubation of 45 minutes at 15°C., the reaction was terminated by the addition of 30 μl TE followed bysequential phenol/chloroform and chloroform extractions and backextracted with 20 μl TE as described above. The DNA was ethanolprecipitated in the presence of 2 μl Pellet Paint™ (Novagen) carrier and0.3 M sodium acetate and was resuspended 11 μl of water.

Eco RI adapters were ligated onto the 5′ ends of the cDNA describedabove to enable cloning into an expression vector. 11 μl of cDNA and 4μl of 65 pmole/μl of Eco RI hemiphophorylated adaptor (Pharmacia BiotechCorp) were mixed with 5 μl 5× ligase buffer (Life Technologies), 2 μl of10 mM ATP and 3 μl of 1 U/μl T4 DNA ligase (Life Technologies), 1 μl 10×ligation buffer (Promega Corp), 9 μl water. The extra dilution with 1×buffer was to prevent the pellet paint from precipitating. The reactionwas incubated 9 hours in a water bath temperature ramp from 10° C. to22° C. over 9 hours, followed by 45 minutes at 25° C. The reaction wasterminated by incubation at 68° C. for 15 minutes.

To facilitate the directional cloning of the cDNA into an expressionvector, the cDNA was digested with XhoI, resulting in a cDNA having a 5′Eco RI cohesive end and a 3′ XhoI cohesive end. The XhoI restrictionsite at the 3′ end of the cDNA had been previously introduced using theZC18698 (SEQ ID NO:27) primer. Restriction enzyme digestion was carriedout in a reaction mixture containing 35 μl of the ligation mix describedabove, 6 μl of 10× H buffer (Boehringer Mannheim Corp.), 1 μl of 2 mg/mlBSA (Biolabs Corp.), 1.7 μl water and 1.0 μl of 40 U/μl XhoI (BoehringerMannheim). Digestion was carried out at 37° C. for 1 hour. The reactionwas terminated by incubation at 68° C. for 15 minutes followed byethanol precipitation, washing drying as described above andresuspension in 30 μl water.

The resuspended cDNA was heated to 65° C. for 5 minutes and cooled onice, 4 μl of 5× gel loading dye (Research Genetics Corp.) was added, thecDNA was loaded onto a 0.8% low melt agarose 1× TAE gel (SEA PLAQUE GTG™low melt agarose; FMC Corp.) and electrophoresed. The contaminatingadapters and cDNA below 0.6 Kb in length were excised from the gel. Theelectrodes were reversed, molten agarose was added to fill in the wells,the buffer was changed and the cDNA was electrophoresed untilconcentrated near the lane origin. The area of the gel containing theconcentrated cDNA was excised and placed in a microfuge tube, and theagarose was melted by heating to 65° C. for 15 minutes. Followingequilibration of the sample to 45° C., 2 μl of 1 U/μl Beta-agarase I(Biolabs, Inc.) was added, and the mixture was incubated for 90 min. at45° C. to digest the agarose. After incubation, 1 tenth volume of 3 M Naacetate was added to the sample, and the mixture was incubated on icefor 15 minutes. The sample was centrifuged at 14,000×g for 15 minutes atroom temperature to remove undigested agarose, the cDNA was ethanolprecipitated, washed in 70% ethanol, air-dried and resuspended in 40 μlwater.

To determine the optimum ratio of cDNA to vector several ligations wereassembled and electroporated. Briefly, 2 μl of 5×T4 ligase buffer(LifeTechnologies), 1 μl of 10 mM ATP, 1 μl pZP7NX digested with EcoR1-Xho1,1 ll T4 DNA ligase diluted to 0.25 u/μl (Life Technologies) water to 10μl and 0.5, 1,2 or 3 μl of cDNA were mixed in 4 separate ligations,incubated at 22° C. for 4 hours, 68° C. for 20 minutes, sodiumacetate-ethanol precipitated, washed, dried and resuspended in 10 ll. Asingle microliter of each ligation was electroporated into 40 μl DH10bElectroMax™ electrocompetent bacteria (Life Technologies) using a 0.1 cmcuvette (Biorad) and a Genepulser, pulse controller™ (Biorad) set to 2.5KV, 251 F, 200 ohms. These cells were immediately resuspended in 1 ml.SOC broth (Manniatis, et al. supra.) followed by 500 ll of 50%glycerol-SOC as a preservative. These “glycerol stocks” were frozen inseveral aliquots at −70° C. An aliquot of each was thawed and platedserially on LB-agar plates supplemented with ampicillin at 100 μg/ml.Colony numbers indicated that the optimum ratio of CD3+ cDNA to pZP7NXvector was 1 μl to 45 ng; such a ligation yielded 4.5 million primaryclones.

For the purpose of screening this library using a BaF3-zalpha11 basedproliferation assay (Example 5) glycerol stocks from above were dilutedinto liquid cultures of 100 or 250 clones per pool in deep wellmicrotiter plates, grown 24 hours at 37° C. with shaking and plasmidisolated using a Qiagen kit following the manufacturer's instructions.Such DNA was subsequently transfected into BHK cells, media conditioned72 hours, harvested and placed on 5K BaF3-zalpha11 cells for 72 hoursafter which proliferation was assessed using an “Alamar blue”fluorescence assay (Example 5B and Example 2B)

For the purpose of screening the library by secretion trap cloning, acomplex, amplified form of the library was needed to transfect COS-7cells. About 4.8 million clones were plated on 110 15 cm LB-agar platessupplemented with 100 μg/ml ampicillin, 10 μg/ml methicillin. Aftergrowing the plates overnight at 37° C. the bacteria were harvested byscraping and pelleted. Plasmid DNA was extracted from the pelletedbacteria using a Nucleobond-giga™ (Clonetech) following themanufacturer's instructions. This plasmid was then used to transfectCOS-7 cells (ATCC No. CRL 1651) on slides and screened using thesecretion trap technique described below (Example 12).

Example 7 Expression Cloning of Human Zalpha11 Ligand

The glycerol stocks from the activated human CD3+ selected cell library(Example 6) were added to Super Broth II™ (Becton Dickinson,Cockeysville, Md.)+0.1 mg/ml ampicillin (amp) at a concentration of 250cells per 800 microliters. The E. coli were allowed to equilibrate for24 hours at room temperature. At the time of inoculation, 400microliters was plated on LB +amp plates to determine the actual titerof the inoculation. After 24 hours the plates were counted and then thefinal concentration of the SuperBrothII™+E. coli was adjusted so thatthe final concentration was 250 cells per 1.2 ml. Three times 2 literswere inoculated for a total of 6 liters. The media were then plated into96-well deep well blocks (Qiagen). Plating was done on the 8-channelQ-Fill2™ dispenser (Genetix, Christchurch, Dorset, UK). The E. coli weregrown overnight at 37° C. shaking at 250 rotations/min. on a NewBrunswick Scientific Innova 4900 multi-tier environment shaker. The E.coli were spun out of solution at 3000 rpm, using a Beckman GS-6KRcentrifuge. These E. coli pellets were frozen at −20° C. or used freshbefore miniprepping the plasmid DNA. Each pellet contains approximately250 cDNA clones from the human CD3+ selected cell library.

These pools of 250 cDNA clones were then mini-prepped using QIAprep™ 96Turbo Miniprep kit (Qiagen). Plasmid DNA was eluted using 125 μl of TE(10 mM Tris pH 8, 1 mM EDTA). This plasmid DNA was then used totransfect BHK cells.

BHK Transfection

BHK cells were plated in 96-well tissue culture plates at a density of12,000 cells per well in a volume of 100 μl. per well. Culture media wasDMEM (GibcoBRL), 5% heat-inactivated fetal bovine serum, 2 mML-glutamine (GibcoBRL), 1×PSN (GibcoBRL), 1 mM NaPyruvate (GibcoBRL).

The following day, BHK cells were washed once with 100 μl SFA. SFA isserum-free media which is DMEM/F12 (Gibco/BRL), 2 mM GlutaMax™(Gibco/BRL), 1 mM NaPyruvate, 10 μg/ml transferrin, 5 μg/ml insulin, 10μg/ml fetuin, 2 μg/ml selenium, 25mM HEPES (Gibco/BRL), 100 μMnon-essential amino acids (Gibco/BRL).

A DNA/Lipofectamine™ mix is made as follows: 2.2 μl Lipofectamine™reagent (Gibco/BRL) is combined with 102.8 μl SFA at room temperature;approximately 5 μl of the plasmid DNA (200 ng/μl) is then added to theLipofectamine™/SFA to form the DNA/Lipofectamine™ mixture, which isincubated at room temperature for 30 minutes. The SFA was removed fromthe BHK cells and the cells were incubated with 50 μl of theDNA/lipofectamine™ mix for 5 hours at 37° C. with 5% CO₂. Fifty μl ofthe DNA/Lipofectamine™ mixture was added to each of two wells of the BHKcells, so that transfections were done in duplicate.

After BHK cells were incubated with DNA/Lipofectamine™ mix for 5 hours,the DNA/Lipofectamine™ mix was removed and 100 μl culture media wasadded. Cells were incubated overnight, the media was removed andreplaced with 100 μl. culture media. After culturing cells for 72 hours,conditioned media was removed, frozen at −80° C. for a minimum of 20minutes, thawed, and then 50 μl was assayed in the zalpha11/BaF3proliferation assay, described in Examples 2B and Example 5, to identifypools of 250 clones with ligand activity.

Thirty-five 96-well plates were screened in a single assay. Thisrepresented approximately 250 cDNAs/well or 840,000 cDNAs total. Ofthese, conditioned media from 54 wells (representing 250 cDNAs per well)tested positive in the proliferation assay. The conditioned media fromthese positive pools was re-tested in a second assay (secretion trap)with and without the soluble receptor (see, Example 12). The zalpha11CEEsoluble receptor (Example 10A) was used at a final concentration ofabout 1 μg/ml. For all 54 positive pools, essentially all of theactivity was neutralized by addition of the soluble zalpha11 receptor,indicating that these pools contained a cDNA from the zalpha11 Ligand.Four of these positive pools were chosen to break-down and isolate asingle cDNA that would encode the zalpha11 Ligand. These were 45C5,46G11 , 40H12, and 60A1.

For each of these 4 pools, 1 μl. of DNA was used to transformElectroMax™ DH10B cells (Gibco/BRL) by electroporation. Thetransformants were plated on LB+amp (100 μg/ml)+methicillin (10 μg/ml)plates to give single colonies. For each electroporated pool, 960individual colonies were toothpicked into ten 96-well plates containing1.2 ml of SuperBrothII™ per well. These plates were numbered #1-10 foreach of the breakdown pools (45C5, 46G11, 40H12, and 60A1). These werecultured overnight and the plasmid DNA miniprepped as above. For 46G11,40H12, and 60A1, plasmid DNA from the breakdown plates was transfectedinto BHK cells as above.

For 45 C5, a “fast track” protocol was utilized to accelerate theidentification of the zalpha11 Ligand cDNA. BHK cells were transfectedwith plasmid DNA from the breakdown plates as above, DNA/Lipofectamine™mix was removed after a 5 hour incubation, and culture media was added.Since the transfections were done in duplicate, the culture media washarvested the following day after 24 hours from one of the transfectedBHK plates, and harvested the following day after 48 hours from theremaining transfected plate. The 24 hour conditioned media was assayedas above for zalpha11 Ligand activity using the proliferation assay asdescribed herein.

Plasmid DNA was pooled from 45C5 breakdown plates #1-4 and assayed forbinding of zalpha11 soluble receptor to its ligand by the“secretion-trap” protocol (see, Example 12, below). Eight positiveclones were identified from a total of 384 sequences. Results from theproliferation assay confirmed activity of the zalpha11 Ligand andcorrelated with results of the secretion trap assay (see Example 12).Concurrently, plasmid DNA miniprepped from plates #1-4 of the 45C5 poolbreakdown was sequenced to determine the DNA sequence of each of the 384clones.

Several clones that were positively identified in the proliferation andsecretion trap assays were also sequenced using the following primers:ZC14,063 (SEQ ID NO:28), ZC7,764a (SEQ ID NO:38), ZC7,764b (SEQ IDNO:39), ZC22,034 (SEQ ID NO:40), and ZC22,035 (SEQ ID NO:41). Thepolynucleotide sequence of zalpha11 Ligand was full-length (SEQ ID NO:1)and its corresponding amino acid sequence is shown (SEQ ID NO:2).

Example 8 Construction of Mammalian Expression Vectors that ExpressZalpha11 Soluble Receptors: Zalpha11CEE, Zalpha11CFLG, Zalpha11CHIS andZalpha11-Fc4

A. Construction of Zalpha11 Mammalian Expression Vector ContainingZalpha11CEE, Zalpha11CFLG and Zalpha11CHIS

An expression vector was prepared for the expression of the soluble,extracellular domain of the zalpha11 polypeptide, pC4zalpha11CEE,wherein the construct is designed to express a zalpha11 polypeptidecomprised of the predicted initiating methionine and truncated adjacentto the predicted transmembrane domain, and with a C-terminal Glu-Glu tag(SEQ ID NO:29).

A 700 bp PCR generated zalpha11 DNA fragment was created using ZC19,931(SEQ ID NO:30) and ZC19,932 (SEQ ID NO:31) as PCR primers to add Asp718and BamHI restriction sites. A plasmid containing the zalpha11 receptorcDNA (SEQ ID NO:7) was used as a template. PCR amplification of thezalpha11 fragment was performed as follows: Twenty five cycles at 94 C.for 0.5 minutes; five cycles at 94° C. for 10 seconds, 50° C. for 30seconds, 68° C. for 45 seconds, followed by a 4° C. hold. The reactionwas purified by chloroform/phenol extraction and isopropanolprecipitation, and digested with Asp718 and BamHI (Gibco BRL) followingmanufacturer's protocol. A band of the predicted size, 700 bp, wasvisualized by 1% agarose gel electrophoresis, excised and the DNA waspurified using a QiaexII™ purification system (Qiagen) according themanufacturer's instructions.

The excised DNA was subcloned into plasmid pC4EE which had been cut withBamHI and Asp718. The pC4zalpha11CEE expression vector uses the nativezalpha11 signal peptide and attaches the Glu-Glu tag (SEQ ID NO:29) tothe C-terminus of the extracellular portion of the zalpha11polypeptide-encoding polynucleotide sequence. Plasmid pC4EE, is amammalian expression vector containing an expression cassette having themouse metallothionein-1 promoter, multiple restriction sites forinsertion of coding sequences, a stop codon and a human growth hormoneterminator. The plasmid also has an E. coli origin of replication, amammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a DHFR gene and the SV40 terminator.

About 30 ng of the restriction digested zalpha11 insert and about 12 ngof the digested vector were ligated overnight at 16° C. One microliterof each ligation reaction was independently electroporated into DH10Bcompetent cells (GIBCO BRL, Gaithersburg, Md.) according tomanufacturer's direction and plated onto LB plates containing 50 mg/mlampicillin, and incubated overnight. Colonies were screened byrestriction analysis of DNA prepared from 2 ml liquid cultures ofindividual colonies. The insert sequence of positive clones was verifiedby sequence analysis. A large scale plasmid preparation was done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

The same process was used to prepare the zalpha11 soluble receptors witha C-terminal his tag, composed of 6 His residues in a row; and aC-terminal flag (SEQ ID NO:37) tag, zalpha11CFLAG. To prepare theseconstructs, the aforementioned vector has either the HIS or the FLAG®tag in place of the glu-glu tag (SEQ ID NO:29).

B. Mammalian Expression Construct of Soluble Zalpha11 ReceptorZalpha11-Fc4

An expression plasmid containing all or part of a polynucleotideencoding zalpha11 was constructed via homologous recombination. Theextracellular domain of the zalpha11 receptor was fused to the Fc regionderived from human IgG, called “Fc4” (SEQ ID NO:33) which contains amutation so that it no longer binds the Fc receptor. A fragment ofzalpha11 cDNA was isolated using PCR that includes the polynucleotidesequence from extracellular domain of the zalpha11 receptor. The twoprimers used in the production of the zalpha11 fragment were: (1) Theprimers for PCR each include from 5′ to 3′ end: 40 bp of the vectorflanking sequence (5′ of the insert) and 17 bp corresponding to the 5′end of the zalpha11 extracellular domain (SEQ ID NO:32); and (2) 40 bpof the 5′ end of the Fc4 polynucleotide sequence (SEQ ID NO:33) and 17bp corresponding to the 3′ end of the zalpha11 extracellular domain (SEQID NO:34). The fragment of Fc-4 for fusion with the zalpha11 wasgenerated by PCR in a similar fashion. The two primers used in theproduction of the Fc4 fragment were: (1) a 5′ primer consisting of 40 bpof sequence from the 3′ end of zalpha11 extracellular domain and 17 bpof the 5′ end of Fc4 (SEQ ID NO:35); and (2) a 3′ primer consisting of40 bp of vector sequence (3′ of the insert) and 17 bp of the 3′ end ofFc4 (SEQ ID NO:36).

PCR amplification of the each of the reactions described above wasperformed as follows: one cycle at 94° C. for 2 minutes; twenty-fivecycles at 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1minute; one cycle at 72° C. for 5 minutes; followed by a 4° C. hold. Tenμl of the 100 μl PCR reaction was run on a 0.8% LMP agarose gel(Seaplaque GTG) with 1×TBE buffer for analysis. The remaining 90 μl ofthe PCR reaction is precipitated with the addition of 5 μl 1 M NaCl and250 μl of absolute ethanol. The expression vector used was derived fromthe plasmid pCZR199 derived from pZP9 (ATCC Deposit No. 98668), and wascut with SmaI (BRL). The expression vector was derived from the plasmidpCZR199, and is a mammalian expression vector containing an expressioncassette having the CMV immediate early promoter, a consensus intronfrom the variable region of mouse immunoglobulin heavy chain locus,multiple restriction sites for insertion of coding sequences, a stopcodon and a human growth hormone terminator. The expression vector alsohas an E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene and the SV40 terminator. The expression vectorused was constructed from pCZR199 by the replacement of themetallothionein promoter with the CMV immediate early promoter.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl containing approximately 1 μg each of the zalpha11and Fc4 inserts, and 100 ng of SmaI (BRL) digested expression vector andtransferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixtureswere electropulsed at 0.75 kV (5 kV/cm), “infinite” ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol and the yeast was plated intwo 300 μl aliquots onto two URA-D plates and incubated at 30° C.

After about 48 hours, the Ura+ yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 1 ml of lysis buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) isdone with 0.5-2 ml yeast DNA prep and 40 μl of DH10B cells. The cellswere electropulsed at 2.0 kV, 25 mF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) was plated in 250 μl aliquots on four LB AMPplates (LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/LAmpicillin).

Individual clones harboring the correct expression construct forzalpha11-Fc4 were identified by restriction digest to verify thepresence of the zalpha11 -Fc4 insert and to confirm that the various DNAsequences have been joined correctly to one another. The insert ofpositive clones were subjected to sequence analysis. Larger scaleplasmid DNA is isolated using the Qiagen Maxi kit (Qiagen) according tomanufacturer's instructions.

Example 9 Transfection And Expression Of Zalpha11 Soluble ReceptorPolypeptides

A. Mammalian Expression of Soluble Zalpha11 Receptor Zalpha11CEE,Zalpha11CFLG, and Zalpha11CHIS

BHK 570 cells (ATCC No. CRL-10314), passage 27, were plated at 1.2×10⁶cells/well (6-well plate) in 800 μl of serum free (SF) DMEM media (DMEM,Gibco/BRL High Glucose) (Gibco BRL, Gaithersburg, Md.). The cells weretransfected with expression plasmids containing zalpha11CEE,zalpha11CFLG, or zalpha11CHIS described above (see, Example 8), usingLipofectin™ (Gibco BRL), in serum free (SF) DMEM. Three micrograms ofzalpha11CEE, zalpha11CFLG, or zalpha11CHIS each were separately dilutedinto 1.5 ml tubes to a total final volume of 100 μl SF DMEM. In separatetubes, 15 μl of Lipofectin™ (Gibco BRL) was mixed with 100 μl of SFDMEM. The Lipofectin™ mix was incubated at room temperature for 30-45minutes then the DNA mix was added and allowed to incubate approximately10-15 minutes at room temperature.

The entire DNA: Lipofectin™ mixture was added to the plated cells anddistributed evenly over them. The cells were incubated at 37° C. forapproximately five hours, then transferred to separate 150 mm MAXIplates in a final volume of 30 ml DMEM/5% fetal bovine serum (FBS)(Hyclone, Logan, Utah). The plates were incubated at 37° C., 5% CO₂,overnight and the DNA: Lipofectin™ mixture was replaced with selectionmedia (5% FBS/DMEM with 1 μM methotrexate (MTX))the next day.

Approximately 10-12 days post-transfection, the plates were washed with10 ml SF DMEM. The wash media was aspirated and replaced with 7.25 mlserum-free DMEM. Sterile Teflon meshes (Spectrum Medical Industries, LosAngeles, Calif.) pre-soaked in SF DMEM were then placed over the clonalcell colonies. A sterile nitrocellulose filter pre-soaked in SF DMEM wasthen placed over the mesh. Orientation marks on the nitrocellulose weretransferred to the culture dish. The plates were then incubated for 5-6hours in a 37° C., 5% CO₂ incubator.

Following incubation, the filters/meshes were removed, and the mediaaspirated and replaced with 5% FBS/DMEM with 1 μM MTX. The filters werethen blocked in 10% nonfat dry milk/Western A buffer (Western A: 50 mMTris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and 0.25% gelatin) for15 minutes at room temperature on a rotating shaker. The filters werethen incubated with an anti-Glu-Glu, anti-FLAG®, or anti-HISantibody-HRP conjugates, respectively, in 2.5% nonfat dry milk/Western Abuffer for one hour at room temperature on a rotating shaker. Thefilters were then washed three times at room temperature with Western Afor 5-10 minutes per wash. The filters were developed with ultra ECLreagent (Amersham Corp., Arlington Heights, Ill.) according themanufacturer's directions and visualized on the Lumi-Imager (RocheCorp.)

Positive expressing clonal colonies were mechanically picked to 12-wellplates in one ml of 5% FCS/DMEM with 5 μM MTX, then grown to confluence.Conditioned media samples were then tested for expression levels viaSDS-PAGE and Western anlaysis. The three highest expressing clones foreach construct were picked; two out of three were frozen down as back upand one was expanded for mycoplasma testing and large-scale factoryseeding.

B. Mammalian Expression of Soluble Zalpha11 Receptor Zalpha11-Fc4

BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm tissue culturedishes and allowed to grow to approximately 50 to 70% confluencyovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone,Logan, Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mMsodium pyruvate (Gibco BRL)). The cells were then transfected with theplasmid containing zalpha11-Fc4 (see, Example 8), using Lipofectamine™(Gibco BRL), in serum free (SF) media formulation (DMEM, 10 mg/mltransferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1%sodium pyruvate). The plasmid containing zalpha11-Fc4 was diluted into15 ml tubes to a total final volume of 640 ml with SF media. 35 ml ofLipofectamine™ (Gibco BRL) was mixed with 605 ml of SF medium. TheLipofectamine™ mix was added to the DNA mix and allowed to incubateapproximately 30 minutes at room temperature. Five milliliters of SFmedia was added to the DNA:Lipofectamine™ mixture. The cells were rinsedonce with 5 ml of SF media, aspirated, and the DNA:Lipofectamine™mixture is added. The cells were incubated at 37° C. for five hours,then 6.4 ml of DMEM/10% FBS, 1% PSN media was added to each plate. Theplates were incubated at 37° C. overnight and the DNA:Lipofectamine™mixture was replaced with fresh 5% FBS/DMEM media the next day. On day 2post-transfection, the cells were split into the selection media(DMEM/FBS media from above with the addition of 1 μM methotrexate (SigmaChemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50.The media on the cells was replaced with fresh selection media at day 5post-transfection. Approximately 10 days post-transfection, two 150 mmculture dishes of methotrexate resistant colonies from each transfectionwere trypsinized and the cells are pooled and plated into a T-162 flaskand transferred to large scale culture.

Example 10 Purification of Zalpha11 Soluble Receptors from BHK 570 Cells

A. Purification of Zalpha11CEE Polypeptide from BHK 570

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying zalpha11 polypeptidecontaining C-terminal GluGlu (EE) tags. Thirty liters of cell factoryconditioned media was concentrated to 1.6 liters with an Amicon S10Y3spiral cartridge on a ProFlux A30. A Protease inhibitor solution wasadded to the concentrated 1.6 liters of cell factory conditioned mediafrom transfected BHK 570 cells (see, Example 9) to final concentrationsof 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). Samples were removed for analysis and the bulkvolume was frozen at −80° C. until the purification was started. Totaltarget protein concentrations of the concentrated cell factoryconditioned media was determined via SDS-PAGE and Western blot analysiswith the anti-EE HRP conjugated antibody.

A 100 ml column of anti-EE G-Sepharose (prepared as described below) waspoured in a Waters AP-5, 5 cm×10 cm glass column. The column was flowpacked and equilibrated on a BioCad Sprint (PerSeptive BioSystems,Framingham, Mass.) with phosphate buffered saline (PBS) pH 7.4. Theconcentrated cell factory conditioned media was thawed, 0.2 micronsterile filtered, pH adjusted to 7.4, then loaded on the columnovernight with 1 ml/minute flow rate. The column was washed with 10column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4), thenplug eluted with 200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide(Anaspec, San Jose, Calif.) at 5 ml/minute. The EE peptide used has thesequence EYMPME (SEQ ID NO:29). The column was washed for 10 CVs withPBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The pH of theglycine-eluted column was adjusted to 7.0 with 2 CVs of 5×PBS, thenequilibrated in PBS (pH 7.4). Five ml fractions were collected over theentire elution chromatography and absorbance at 280 and 215 nM weremonitored; the pass through and wash pools were also saved and analyzed.The EE-polypeptide elution peak fractions were analyzed for the targetprotein via SDS-PAGE Silver staining and Western Blotting with theanti-EE HRP conjugated antibody. The polypeptide elution fractions ofinterest were pooled and concentrated from 60 ml to 5.0 ml using a10,000 Dalton molecular weight cutoff membrane spin concentrator(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate zalpha11CEE from other co-purifying proteins, theconcentrated polypeptide elution pooled fractions were subjected to aPOROS HQ-50 (strong anion exchange resin from PerSeptive BioSystems,Framingham, Mass.) at pH 8.0. A 1.0×6.0 cm column was poured and flowpacked on a BioCad Sprint. The column was counter ion charged thenequibrated in 20 mM TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). Thesample was diluted 1:13 (to reduce the ionic strength of PBS) thenloaded on the Poros HQ column at 5 ml/minute. The column was washed for10 CVs with 20 mM Tris pH 8.0 then eluted with a 40 CV gradient of 20 mMTris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions werecollected over the entire chromatography and absorbance at 280 and 215nM were monitored. The elution peak fractions were analyzed via SDS-PAGESilver staining. Fractions of interest were pooled and concentrated to1.5-2 ml using a 10,000 Dalton molecular weight cutoff membrane spinconcentrator (Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate zalpha11CEE polypeptide from free EE peptide and anycontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to chromatography on a 1.5×90 cm Sephadex S200(Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBS at aflow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractions werecollected across the entire chromatography and the absorbance at 280 and215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified zalpha11CEE polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox(Sterogene) column to remove any remaining endotoxins. The sample waspassed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

On Western blotted, Coomassie Blue and Silver stained SDS-PAGE gels, thezalpha11CEE polypeptide was one major band of an apparent molecularweight of about 50,000 Daltons. The mobility of this band was the sameon reducing and non-reducing gels.

The protein concentration of the purified material was performed by BCAanalysis (Pierce, Rockford, Ill.) and the protein was aliquoted, andstored at −80° C. according to our standard procedures. On IEF(isoelectric focusing) gels the protein runs with a PI of less than 4.5.The concentration of zalpha11CEE polypeptide was 1.0 mg/ml.

To prepare anti-EE Sepharose, a 100 ml bed volume of protein G-Sepharose(Pharmacia, Piscataway, N.J.) was washed 3 times with 100 ml of PBScontaining 0.02% sodium azide using a 500 ml Nalgene 0.45 micron filterunit. The gel was washed with 6.0 volumes of 200 mM triethanolamine, pH8.2 (TEA, Sigma, St. Louis, Mo.), and an equal volume of EE antibodysolution containing 900 mg of antibody was added. After an overnightincubation at 4° C., unbound antibody was removed by washing the resinwith 5 volumes of 200 mM TEA as described above. The resin wasresuspended in 2 volumes of TEA, transferred to a suitable container,and dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in TEA,was added to a final concentration of 36 mg/ml of protein G-Sepharosegel. The gel was rocked at room temperature for 45 min and the liquidwas removed using the filter unit as described above. Nonspecific siteson the gel were then blocked by incubating for 10 min. at roomtemperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gelwas then washed with 5 volumes of PBS containing 0.02% sodium azide andstored in this solution at

B. Purification of Zalpha11CFLAG polypeptide from BHK 570

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying zalpha11 polypeptidecontaining C-terminal FLAG® (FLG) (Sigma-Aldrich Co.) tags. Thirtyliters of cell factory conditioned media was concentrated to 1.7 literswith an Amicon S10Y3 spiral catridge on a ProFlux A30. A Proteaseinhibitor solution was added to the 1.7 liters of concentrated cellfactory conditioned media from transfected BHK 570 cells (see, Example9) to final concentrations of 2.5 mM ethylenediaminetetraacetic acid(EDTA, Sigma Chemical Co. St. Louis, Mo.), 0.003 mM leupeptin(Boehringer-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin(Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). Sampleswere removed for analysis and the bulk volume was frozen at −80° C.until the purification was started. Total target protein concentrationsof the cell factory conditioned media was determined via SDS-PAGE andWestern blot analysis with the anti-FLAG® (Kodak) HRP conjugatedantibody. A 125 ml column of anti-FLAG® M2-Agarose affinity gel(Sigma-Aldrich Co.) was poured in a Waters AP-5, 5 cm×10 cm glasscolumn. The column was flow packed and equilibrated on a BioCad Sprint(PerSeptive BioSystems, Framingham, Mass.) with phosphate bufferedsaline (PBS) pH 7.4. The concentrated cell factory conditioned media wasthawed, 0.2 micron sterile filtered, pH adjusted to 7.4, then loaded onthe column overnight with 1 ml/minute flow rate. The column was washedwith 10 column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4),then plug eluted with 250 ml of PBS (pH 6.0) containing 0.5 mg/ml FLAG®(Sigma-Aldrich Co.) peptide at 5 ml/minute. The FLAG® peptide used hasthe sequence DYKDDDDK (SEQ ID NO:37). The column was washed for 10 CVswith PBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The pH of theglycine-eluted column was adjusted to 7.0 with 2 CVs of 5×PBS, thenequilibrated in PBS (pH 7.4). Five ml fractions were collected over theentire elution chromatography and absorbence at 280 and 215 nM weremonitored; the pass through and wash pools were also saved and analyzed.The FLAG®-polypeptide elution peak fractions were analyzed for thetarget protein via SDS-PAGE Silver staining and Western Blotting withthe anti-FLAG HRP conjugated antibody. The polypeptide elution fractionsof interest were pooled and concentrated from 80 ml to 12 ml using a10,000 Dalton molecular weight cutoff membrane spin concentrator(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate zalpha11CFLG from other co-purifying proteins, thepolypeptide elution pooled fractions were subjected to a POROS HQ-50(strong anion exchange resin from PerSeptive BioSystems, Framingham,Mass.) at pH 8.0. A 1.0×6.0 cm column was poured and flow packed on aBioCad Sprint. The column was counter ion charged then equilibrated in20 mM TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). The sample wasdiluted 1:13 (to reduce the ionic strength of PBS) then loaded on thePoros HQ-50 column at 5 ml/minute. The column was washed for 10 columnvolumes (CVs) with 20 mM Tris pH 8.0 then eluted with a 40 CV gradientof 20 mM Tris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 mlfractions were collected over the entire chromatography and absorbanceat 280 and 215 nM were monitored. The elution peak fractions wereanalyzed via SDS-PAGE Silver staining. Fractions of interest were pooledand concentrated to 1.5-2 ml using a 10,000 Dalton molecular weightcutoff membrane spin concentrator (Millipore, Bedford, Mass.) accordingto the manufacturer's instructions.

To separate zalpha11CFLG polypeptide from free FLAG® peptide and anycontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to chromatography on a 1.5×90 cm Sephacryl S200(Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBS at aflow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractions werecollected across the entire chromatography and the absorbance at 280 and215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified zalpha11CFLG polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox(Sterogene) column to remove any remaining endotoxins. The sample waspassed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

On Western blotted, Coomassie Blue and Silver stained SDS-PAGE gels, thezalpha11CFLG polypeptide was one major band of an apparent molecularweight of about 50,000 Daltons. The mobility of this band was the sameon reducing and non-reducing gels.

The protein concentration of the purified material was performed by BCAanalysis (Pierce, Rockford, Ill.) and the protein was aliquoted, andstored at −80° C. according to our standard procedures. On IEF(isoelectric focusing) gels the protein runs with a PI of less than 4.5.The concentration of zalpha11CFLG polypeptide was 1.2 mg/ml.

C. Purification of Zalpha11-Fc4 Polypeptide from Transfected BHK 570Cells

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying zalpha11 polypeptidecontaining C-terminal fusion to human IgG/Fc (zalpha11-Fc4; Examples 8and 9). 12,000 ml of conditioned media from BHK 570 cells transfectedwith zalpha11-Fc4 (Example 9) was filtered through a 0.2 mm sterilizingfilter and then supplemented with a solution of protease inhibitors, tofinal concentrations of, 0.001 mM leupeptin (Boerhinger-Mannheim,Indianapolis, Ind.), 0.001 mM pepstatin (Boerhinger-Mannheim) and 0.4 mMPefabloc (Boerhinger-Mannheim). A protein G sepharose (6 ml bed volume,Pharmacia Biotech) was packed and washed with 500 ml PBS (Gibco/BRL) Thesupplemented conditioned media was passed over the column with a flowrate of 10 ml/minute, followed by washing with 1000 ml PBS (BRL/Gibco).zalpha11-Fc4 was eluted from the column with 0.1 M Glycine pH 3.5 and 2ml fractions were collected directly into 0.2 ml 2M Tris pH 8.0, toadjust the final pH to 7.0 in the fractions.

The eluted fractions were characterized by SDS-PAGE and western blottingwith anti-human Fc (Amersham) antibodies. Western blot analysis ofreducing SDS-PAGE gels reveal an immunoreactive protein of about 80,000KDa in fractions 2-10. Silver stained SDS-PAGE gels also revealed an80,000 KDa zalpa11:Fc polypeptide in fractions 2-10. Fractions 2-10 werepooled.

The protein concentration of the pooled fractions was performed by BCAanalysis (Pierce, Rockford, Ill.) and the material was aliquoted, andstored at −80° C. according to our standard procedures. Theconcentration of the pooled fractions was 0.26 mg/ml.

Example 11 Assay Using Zalpha11 Soluble Receptor Zalpha11CEE,Zalpha11CFLG and Zalpha11-Fc4 (Mutant) Soluble Receptors in CompetitiveInhibition Assay

BaF3/Zalpha11 cells were spun down and washed in mIL-3 free media. Thecells were spun and washed 3 times to ensure the removal of the mIL-3.Cells were then counted in a hemacytometer. Cells were plated in a96-well format at 5000 cells per well in a volume of 100 μl per wellusing the mIL-3 free media.

Both conditioned media from the monkey spleen cell activation and thehuman CD3+ selected cells, described in Example 5, were added inseparate experiments at 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and0.375% concentrations, with or without zalpha11 soluble receptors (CEE,C-flag, and Fc4 constructs; See, Example 9 and 10) at 10 μg/ml. Thetotal assay volume was 200 μl.

The assay plates were incubated 37° C., 5% CO₂ for 3 days at which timeAlamar Blue (Accumed) was added at 20 μl/well. Plates were againincubated at 37° C., 5% CO₂ for 24 hours. Plates were read on the Fmax™plate reader (Molecular Devices) as described in Example 2. Resultsdemonstrated complete inhibition of cell growth from each of thedifferent zalpha11 soluble receptor constructs at 10 μg/ml, confirmingthat the factor in each sample was specific for the zalpha11 receptor.

Titration curves, diluting out the soluble receptors, were also runusing the above stated assay. Both the zalpha11CEE and zalpha11CFLGsoluble zalpha11 receptors were able to completely inhibit growth atconcentrations as low as 20 ng/ml. The mutant zalpha11-Fc4 solublezalpha11 receptor was only as effective at 1.5 μg/ml.

Example 12 Secretion Trap Assay

A secretion trap assay was used to identify the cDNA by for the zalpha11Ligand. The positive DNA pools obtained from the expression cloningeffort described in Example 7.

The DNA pools of 250 clones were transfected into BHK cells in 96-wellformat, and the condition medium were put into the proliferation assayusing BaF3/zalpha11 cells described in Examples 4 and Example 5. SeveralDNA pools gave positive activities which were repeated and neutralizedwith zalpha11 soluble receptors (see Example 11).

One of the positive DNA pools, 45C5, was transfected into COS cells in12-well format, using the Lipofectamine™ method described below. Asecretion trap assay was then performed using zalpha11 soluble receptors(C-terminal Glu-Glu tagged either with or without biotinylation;C-terminal Flag tagged; or Fc4 zalpha11 soluble receptor fusions)(Example 9) to test the direct binding between potential ligand ofzalpha11 receptor in pool 45C5 and zalpha11 soluble receptor (seebelow). The result was positive. Thus, the DNA of pool 45C5 waselectroporated into E. coli, and single colonies were picked into ten96-well plates. Plates were shaken at 37° C. for 24 hours, and then DNAminipreps (QiaPrep™ 96 Turbo Miniprep Kit; Qiagen) were prepared in96-well format using a TomTech Quadra 9600. The plasmid DNA was thenpooled in the format of rows and columns, transfected into COS cells,and then the positive pools were determined by secretion trap asdescribed below.

COS Cell Transfections

The COS cell transfection was performed as follows: Mix 3 ul pooled DNAand 5 ul Lipofectamine™ in 92 ul serum free DMEM media (55 mg sodiumpyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 μgselenium and 5 mg fetuin in 500 ml DMEM), incubate at room temperaturefor 30 minutes and then add 400 ul serum free DMEM media. Add this 500ul mixture onto 1.5×10⁵ COS cells/well plated on 12-well tissue cultureplate and incubate for 5 hours at 37 ° C. Add 500 ul 20% FBS DMEM media(100 ml FBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 mlDMEM) and incubate overnight.

Secretion Trap Assay

The secretion trap was performed as follows: Media was rinsed off cellswith PBS and then fixed for 15 minutes with 1.8% Formaldehyde in PBS.Cells were then washed with TNT (0.1M Tris-HCL, 0.15M NaCl, and 0.05%Tween-20 in H2O), and permeated with 0.1% Triton-X in PBS for 15minutes, and again washed with TNT. Cells were blocked for 1 hour withTNB (0.1M Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NENRenaissance TSA-Direct Kit) in H2O), and washed again with TNT. If usingthe biotinylated protein, the cells were blocked for 15 minuteincubations with Avidin and then Biotin (Vector Labs) washing in-betweenwith TNT. Depending on which soluble receptor was used, the cells wereincubated for 1 hour with: (A) 1-3 μg/ml zalpha11 soluble receptorzalpha11-Fc4 fusion protein (Example 10); (B) 3 μg/ml zalpha11 solublereceptor C-terminal FLAG tagged, zalpha11CFLG (Example 10); (C) 3 μg/mlzalpha11 soluble receptor C-terminal GluGlu tagged, zalpha11CEE (Example10); or (D) 3 μg/ml biotinylated zalpha11 soluble receptor zalpha11CEEin TNB. Cells were then washed with TNT. Depending on which solublereceptor was used, cells were incubated for another hour with: (A) 1:200diluted goat-anti-human Ig-HRP (Fc specific); (B) 1:1000 diluted M2-HRP;(C) 1:1000 diluted anti-GluGlu antibody-HRP; or (D) 1:300 dilutedstreptavidin-HRP (NEN kit) in TNB. Again cells were washed with TNT.

Positive binding was detected with fluorescein tyramide reagent diluted1:50 in dilution buffer (NEN kit) and incubated for 4-6 minutes, andwashed with TNT. Cells were preserved with Vectashield Mounting Media(Vector Labs Burlingame, Calif.) diluted 1:5 in TNT. Cells werevisualized using a FITC filter on fluorescent microscope.

Example 13 Chromosomal Assignment and Placement of the Gene for theZalpha11 Ligand.

The gene for the zalpha11 Ligand was mapped to chromosome 4 using thecommercially available version of the “Stanford G3 Radiation HybridMapping Panel” (Research Genetics, Inc., Huntsville, Ala.). The“Stanford G3 RH Panel” contains DNAs from each of 83 radiation hybridclones of the whole human genome, plus two control DNAs (the RM donorand the A3 recipient). A publicly available WWW server(http://shgc-www.stanford.edu) allows chromosomal localization ofmarkers.

For the mapping of the zalpha11 Ligand gene with the “Stanford G3 RHPanel”, 20 μl reactions were set up in a 96-well microtiter plate(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96”thermal cycler (Stratagene). Each of the 85 PCR reactions consisted of 2μl 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC 22,050 (SEQ ID NO:42), 1 μl antisenseprimer, ZC 22,051 (SEQ ID NO:43), 2 μl “RediLoad” (Research Genetics,Inc., Huntsville, Ala.), 0.4 μl 50× Advantage KlenTaq Polymerase Mix(Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybridclone or control and ddH₂O for a total volume of 20 μl. The reactionswere overlaid with an equal amount of mineral oil and sealed. The PCRcycler conditions were as follows: an initial 1 cycle 5 minutedenaturation at 94° C., 35 cycles of a 45 seconds denaturation at 94°C., 45 seconds annealing at 60° C. and 1 minute AND 15 seconds extensionat 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C.The reactions were separated by electrophoresis on a 2% agarose gel(Life Technologies, Gaithersburg, Md.).

The results showed linkage of the zalpha11 Ligand gene to the IL2framework marker SHGC-12342 with a LOD score of >12 and at a distance of6 cR_(—)10000 (approximately 180 kb) from the marker. The use ofsurrounding markers positions the zalpha11 Ligand gene in the 4q27region on the integrated LDB chromosome 4 map (The Genetic LocationDatabase, University of Southhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html/) .

Example 14 Identification and Cloning of Murine Zalpha11 Ligand Using anEST Sequence to Obtain Full-length Murine Zalpha11 Ligand

A. EST Sequence of Mouse Zalpha11 Ligand

By searching the database with human zalpha11 Ligand cDNA sequence (SEQID NO:1) as a query, a mouse EST (EST1483966) was identified aspotential partial sequence for mouse zalpha11 Ligand. The EST1483966represents a mouse genomic fragment, in which a peptide sequence derivedfrom two potential exons shared high sequence identity with a peptidesegment of the human zalpha11 Ligand (amino acid 13 (Ile) through aminoacid 80 (Gin) of the SEQ ID NO:2).

B. PCR Screen of Mouse Marathon cDNA Panel

Eleven mouse Marathon cDNA (Clontech) samples were screened by PCRdescribed below. The mouse marathon cDNA samples were prepared frombrain, pancreas, kidney, placenta, salivary gland, skin, testis, uterus,embryo, and spleen tissues. They were made in-house using a Marathon™cDNA Amplification Kit (Clontech) according to manufacturer'sinstructions. Based on the EST sequence, two PCR primers, ZC22,056 (SEQID NO:44) and ZC22,057 (SEQ ID NO:45) were used to identify a source ofmouse zalpha11 Ligand by PCR. The PCR reaction conditions were asfollows: 94° C. for 2 min.; 35 cycles at 94° C. for 30 sec., 68° C. for2 min. followed by 68° C. for 4 min.; then a 10° C. soak. The PCRproducts were run on a 1% agarose gel. A strong 150 bp band representingan amplified cDNA fragment was visualized. This indicated mouse spleenmarathon cDNA is the source for mouse zalpha11 Ligand cDNA cloning. Themouse spleen marathon cDNA contained a positive cDNA which wassubsequently identified by sequence analysis as a partial cDNA for mousezalpha11 Ligand.

C. A Composite Sequence for Mouse Full-Length cDNA was Generated by 5′-and 3′-RACE

The 5′ and 3′ flanking sequences of the mouse zalpha11 Ligand partialcDNA sequence were obtained by 5′ and 3′ RACE amplification. Two roundsof nested PCR amplification were performed with additional gene-specificoligo primers ZC22,205 (SEQ ID NO:46) and ZC22,206 (SEQ ID NO:47),ZC22,056 (SEQ ID NO:44) and ZC22,057 (SEQ ID NO:45), and two adapteroligo primers ZC9,739 (SEQ ID NO:48) and ZC9,719 (SEQ ID NO:49). The PCRreactions were run as follows: 94° C. for 2 min; 35 cycles at 94° C. for30 sec, 68° C. for 2 min; followed by 68° C. for 4 min; then a 10° C.soak. The PCR products were run on a 1% agarose gel, and anapproximately 300 bp 5′ RACE product and an approximately 800 bp 3′ RACEproduct were identified. These fragments were isolated using Qiaquick™gel extraction kit (Qiagen).

The purified PCR products were sequenced using the following primers:ZC9,719 (SEQ ID NO:49), ZC22,205 (SEQ ID NO:46) and ZC22,206 (SEQ IDNO:47). A preliminary composite full-length mouse zalpha11 Ligandsequence was identified by combining the 5′ and 3′ RACE fragments. Thefull length mouse clone was isolated as described in Example 15 below.

Example 15 Isolation of Mouse Zalpha11 cDNA Clone from an ActivatedMouse Spleen Library

A. Murine Primary Source Used to Isolate Mouse Zalpha11 Ligand

Mouse spleens from Balb/C mice, were collected and mashed betweenfrosted-end slides to create a cell suspension. The isolated primarymouse cell yield was 6.4×10⁸ cells prior to selection described below.

The spleen cells were suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA, 2mM EDTA). 1.6 ml of cell suspension was removed and 0.4 ml CD90 (Thy1.2)microbeads (Miltenyi Biotec) added. The mixture was incubated for 15min. at 4° C. These cells labeled with CD90 beads were washed with 30 mlMACS buffer, and then resuspended in 2 ml MACS buffer.

A VS+ column (Miltenyi) was prepared according to the manufacturer'sinstructions. The VS+ column was then placed in a VarioMACS™ magneticfield (Miltenyi). The column was equilibrated with 5 ml MACS buffer. Theisolated primary mouse cells were then applied to the column. The CD3negative cells were allowed to pass through. The column was rinsed with9 ml (3×3 ml) MACS buffer. The column was then removed from the magnetand placed over a 15 ml falcon tube. CD90+ cells were eluted by adding 5ml MACS buffer to the column and bound cells flushed out using theplunger provided by the manufacturer. The incubation of the cells withthe CD90 magnetic beads, washes, and VS+ column steps (incubationthrough elution) above were repeated once more. The resulting CD90+fractions from the 2 column separations were pooled. The yield of CD90+selected mouse spleen cells were 1×10⁸ total cells.

A sample of the pooled CD90+ selected mouse cells was removed forstaining and sorting on a fluorescent antibody cell sorter (FACS) toassess their purity. A PE-conjugated hamster anti-mouse CD3ε antibody(PharMingen) was used for staining and sorting the CD90+ selected cells.The mouse CD90+ selected cells were 93% CD3+ cells, suggesting the cellswere 93% T-cells.

The murine CD90+ selected cells were activated by incubating 3×10⁶cells/ml in RPMI+5% FBS+PMA 10 ng/ml and Ionomycin 0.5 μg/ml(Calbiochem) for overnight at 37° C. The supernatant from theseactivated CD90+ selected mouse cells was tested for zalpha11 Ligandactivity as described below. Moreover, the activated CD90+ selectedmouse cells were used to prepare a cDNA library, as described in Example16, below.

Example 16 Cloning of Mouse Zalpha11 Ligand from a Mouse CD90+ SelectedCell Library

Screening of a primary mouse activated CD90+ selected cell cDNA libraryrevealed an isolated cDNA that is a novel member of the four-helixbundle cytokine family. This cDNA encoded the mouse ortholog of thehuman zalpha11 Ligand. The cDNA was identified by hybridizationscreening.

A. The Vector for CD90+ Selected Library Construction

The vector, pZP7N was used for CD3+ selected library construction (SeeExample 6A)

B. Preparation of Primary Mouse Activated CD90+ Selected Cell cDNALibrary

Approximately 1.5×10⁸ primary mouse CD90+ selected cells stimulated 20in ionomycin/PMA (Example 15) were isolated by centrifugation. Total RNAwas isolated from the cell pellet, and converted to double stranded cDNAas described in Example 6B. This DNA was subsequently transfected intoBHK cells, as described in Example 6B, and proliferation was assessedusing an “Alamar blue” fluorescence assay (Example 2B).

For the purpose of screening the library by secretion trap cloning, acomplex, amplified form of the library was needed to transfect COS-7cells. 4.8 million clones were plated on 110 15 cm LB-agar platessupplemented with 100 μg/ml ampicillin, 10 μg/ml methicillin. Aftergrowing the plates overnight at 37° C. the bacteria were harvested byscraping and pelleted. Plasmid DNA was extracted from the pelletedbacteria using a Nucleobond-giga™ (Clonetech) following themanufacturer's instructions. This plasmid was then used to transfectCOS-7 cells on slides and screened using the secretion trap techniquedescribed below (Example 17).

C. Screening the Activated Mouse cDNA Library

Approximately 5×10⁵ clones were plated on 10 LB/Amp Maxi plates. Thecolonies were lifted, denatured, neutralized, and cross-linked using thestandard procedure (Sambrook, J. et al. supra.). Fifty nanograms of the300 bp 5′ RACE PCR fragment (Example 14) was labeled with ³²P usingPrime-Itr RmT random primer labeling kit (Stratagene). The 10 filterswere hybridized with this labeled probe at 65° C. overnight usingExpressHyb™ Hybridization Solution (Clontech). The filters were thenwashed sequentially at 60° C. for 1 hour three times with 0.2×SSC (30 mMNaCl, 3 mM sodium citrate, pH 7.0), 0.1% SDS; and then at 65° C. for 1hour. The filters were exposed at −80° C. overnight, and the X-ray filmwere developed. Agar plugs containing the positive colonies were pulled,and the clones plated on 10-cm LB/Amp plates. The colonies were thenfilter-lifted and hybridized again following the same proceduredescribed above.

One DNA clone, named M11L/pZP7, was isolated and sequenced using thefollowing primers: ZC14,063 (SEQ ID NO:50), ZC5,020 (SEQ ID NO:51),ZC22,421 (SEQ ID NO:52), ZC22,604 (SEQ ID NO:53), and ZC22,641 (SEQ IDNO:54). The polynucleotide sequence of this clone is full-length mousezalpha11 Ligand (SEQ ID NO:55) and consistent with the compositesequence obtained from 5′ and 3′ RACE products. The corresponding aminoacid sequence for the mouse zalpha11 Ligand is shown in SEQ ID NO:56.

Example 17 Mouse Zalpha11 Ligand Binds to Human Zalpha11 SolubleReceptor in Secretion Trap Assay

The DNA for mouse clone M11LpZP7 was transfected into COS cells, and thebinding of human zalpha11 soluble receptor zalpha11-Fc4 (Example 10C) tothe transfected COS cells was tested by a secretion trap assay (Example12). The assay confirmed that the mouse zalpha11 Ligand binds to humanzalpha11 soluble receptor.

The COS cell transfection was performed as per example 12 using 0.7 μgM11U/pZP7 DNA (Example 16) in 3 μl.

The secretion trap was performed as as per example 12 using 1 μg/mlzalpha11 soluble receptor Fc4 fusion protein (Example 10C) in TNB, and1:200 diluted goat-anti-human Ig-HRP (Fc specific) in TNB for thedetectable antibody. Positive binding of the soluble human zalpha11receptor to the prepared fixed cells was detected with fluoresceintyramide reagent as per Example 12. Cells were preserved and visualizedaccording to Example 12.

The positive result indicated the mouse zalpha11 Ligand binds to humanzalpha11 soluble receptor.

Example 18 Expression of Mouse Zalpha11 Ligand in Mammalian Cells

A. Construction of Expression Vector M11L/pZP9

An expression vector was prepared for the expression of the mousezalpha11 Ligand in mammalian cells. A 500 bp PCR generated zalpha11Ligand DNA fragment was created using ZC22,283 (SEQ ID NO:57) andZC22,284 (SEQ ID NO:58) as PCR primers to amplify the coding region ofmouse zalpha11 Ligand and add XhoI and XbaI restriction sites. The mousezalpha11 Ligand clone M11L/pZP7 (Example 16) was used as a template. ThePCR reaction conditions were as follows: 94° C. for 2 min.; 25 cycles at94° C. for 30 sec., 68° C. for 2 min.; followed by 68° C. for 4 min.;then a 10° C. soak. A band of the predicted size, about 500 bp, wasvisualized by 1% agarose gel electrophoresis, excised and the DNA waspurified using a QiaexII™ purification system (Qiagen) according to themanufacturer's instructions. The purified DNA was digested with XhoI andXbaI (Boehringer Mannheim) at 37° C. for 2 hours. Then the DNA was gelisolated and purified following the above procedure.

The excised DNA was subcloned into plasmid pZP9 which was cut with XhoIand XbaI (Boehringer Mannheim). Plasmid pZP9 is a mammalian expressionvector containing an expression cassette having the mousemetallothionein-1 (MT-1) promoter, multiple restriction sites forinsertion of coding sequences, and a human growth hormone terminator.The plasmid also has an E. coli origin of replication, a mammalianselective marker expression unit having an SV40 promoter, enhancer andorigin of replication, a DHFR gene, and the SV40 terminator.

About 30 ng of the restriction digested mouse zalpha11 Ligand fragmentand about 10 ng of the digested pZP9 vector were ligated at roomtemperature for 2 hours. Two μg of ligation reaction was transformedinto INVaF′ competent cells (Invitrogen) according to manufacturer'sprotocol and plated onto LB plates containing 50 μg/ml ampicillin, andincubated at 37° C. overnight. Colonies were screened by restrictionanalysis using XhoI and XbaI (Boerhinger Mannheim) of DNA prepared fromliquid cultures of individual colonies. The insert sequence of positiveclones was verified by sequence analysis to be the mouse zalpha11 Ligandsequence. A large scale plasmid preparation was done using a Qiagen®Maxi prep kit (Qiagen) according to manufacturer's instruction. Theexpression vector that contains mouse zalpha11 Ligand was namedM11L/pZP9.

B. Mammalian Expression of Mouse Zalpha11 Ligand

BHK 570 cells (ATCC No: CRL-10314) were plated in 10 cm tissue culturedishes and allowed to grow to approximately 20% confluence overnight at37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose media;Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone, Logan,Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodiumpyruvate (Gibco BRL)). The cells were then transfected with the plasmidM11LpZP9 (Example 18A) using a mammalian stable CaPO₄ transfection kit(Stratagene) according to the manufacturer's instructions.

One day after transfection, the cells were split 1:10 and 1:20 into theselection media (DMEM/FBS media with the addition of 1 μM methotrexate(Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates. The media on thecells was replaced with fresh selection media at day 5post-transfection. Approximately 10 days post-transfection, methotrexateresistant colonies were trypsinized and the cells pooled and plated intolarge scale culture flasks. Once the cells were grown to approximately90% confluence, they were rinsed with PBS three times, and cultured withserum-free ESTEP2 media (DMEM (Gibco BRL), 0.11 g/l Na Pyruvate, 3.7 g/lNaHCO₃, 2.5 mg/l insulin, 5 mg/l transferrin, pH7.0) conditioned media.The conditioned media were collected three days later, and put into aBaF3 proliferation assay using Alamar Blue, described in Example 19below.

Example 19 Mouse Zalpha11 Ligand Activates Human Zalpha11 Receptor inBaF3 Assay Using Alamar Blue

Proliferation of BaF3/zalpha11 cells (Example 4, and 5B) was assessedusing serum-free conditioned media from BHK cells expressing mousezalpha11 Ligand (Example 18).

BaF3/Zalpha11 cells were spun down, washed and plated in mIL-3 freemedia as described in Example 5B.

Proliferation of the BaF3/Zalpha11 cells was assessed using serum-freeconditioned media from BHK cells expressing mouse zalpha11 Ligand(Example 18). Conditioned media was diluted with mIL-3 free media to:50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations.The proliferation assay was performed as per Example 5B.

Results confirmed the proliferative response of the BaF3/Zalpha11 cellsto mouse zalpha11 Ligand. The response, as measured, was approximately5-fold over background at the 50% concentration.

Example 20 Zalpha11 Ligand Activates Human Zalpha11 Receptor inLuciferase Assay

A. Construction of BaF3/KZ134/Zalpha11 Cell Line

The KZ134 plasmid was constructed with complementary oligonucleotidesZC12,749 (SEQ ID NO:59) and ZC12,748 (SEQ ID NO:60) that contain STATtranscription factor binding elements from 4 genes. A modified c-fos Sisinducible element (m67SIE, or hSIE) (Sadowski, H. et al., Science261:1739-1744, 1993), the p21 SIE1 from the p21 WAF1 gene (Chin, Y. etal., Science 272:719-722, 1996), the mammary gland response element ofthe β-casein gene (Schmitt-Ney, M. et al., Mol. Cell. Biol.11:3745-3755, 1991), and a STAT inducible element of the Fcg RI gene,(Seidel, H. et al., Proc. Natl. Acad. Sci. 92:3041-3045, 1995). Theseoligonucleotides contain Asp718-XhoI compatible ends and were ligated,using standard methods, into a recipient firefly luciferase reportervector with a c-fos promoter (Poulsen, L. K. et al., J. Biol. Chem.273:6229-6232, 1998) digested with the same enzymes and containing aneomycin selectable marker. The KZ134 plasmid was used to stablytransfect BaF3 cells, using standard transfection and selection methods,to make the BaF3/KZ134 cell line.

A stable BaF3/KZ134 indicator cell line, expressing the full-lengthzalpha11 receptor was constructed as per Example 2A, using about 30 μgof the zalpha11 expression vector, described in Example 3. Clones werediluted, plated and selected using standard techniques. Clones werescreened by luciferase assay (see Example 20B, below) using the humanzalpha11 Ligand conditioned media as an inducer. Clones with the highestluciferase response (via STAT luciferase) and the lowest background wereselected. A stable transfectant cell line was selected. The cell linewas called BaF3/KZ134/zalpha11.

B. Human and Mouse Zalpha11 Ligand Activates Human Zalpha11 Receptor inBaF3/KZ134/Zalpha11 Luciferase Assay

BaF3/KZ134/Zalpha11 cells were spun down and washed in mIL-3 free media.The cells were spun and washed 3 times to ensure removal of mIL-3. Cellswere then counted in a hemacytometer. Cells were plated in a 96-wellformat at about 30,000 cells per well in a volume of 100 μl per wellusing the mIL-3 free media. The same procedure was used foruntransfected BaF3/KZ134 cells for use as a control in the subsequentassay.

STAT activation of the BaF3/KZ134/Zalpha11 cells was assessed usingconditioned media from (1) BHK570 cells transfected with the humanzalpha11 Ligand (Example 7) or (2) BHK570 cells transfected with themouse zalpha11 Ligand (Example 18), or (4) mIL-3 free media to measuremedia-only control response. Conditioned media was diluted with RPMImIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and0.375% concentrations. 100 μl of the diluted conditioned media was addedto the BaF3/KZ134/Zalpha11 cells. The assay using the conditioned mediawas done in parallel on untransfected BaF3/KZ134 cells as a control. Thetotal assay volume was 200 μl. The assay plates were incubated at 37°C., 5% CO₂ for 24 hours at which time the cells were pelleted bycentrifugation at 2000 rpm for 10 min., and the media was aspirated and25 μl of lysis buffer (Promega) was added. After 10 minutes at roomtemperature, the plates were measured for activation of the STATreporter construct by reading them on a luminometer (LabsystemsLuminoskan, model RS) which added 40 μl of luciferase assay substrate(Promega) at a five second integration.

Results confirmed the STAT reporter response of the BaF3/KZ134/Zalpha11cells to the human zalpha11 Ligand. The response, as measured, wasapproximately 50 fold over media-only control at the 50% concentration.STAT activation in response to human zalpha11 Ligand was absent in theuntransfected BaF3/KZ134 control cells, showing that the response ismediated through the Zalpha11 receptor.

Results also confirmed the STAT reporter response of theBaF3/KZ134Zalpha11 cells to the mouse zalpha11 Ligand. The response, asmeasured, was approximately 40 fold over media-only control at the 50%concentration. Moreover, STAT activation in response to mouse zalpha11Ligand was evident (about 5-fold) on the untransfected BaF/KZ134 controlcells, suggesting that the murine BaF3 cells may have endogenous mousereceptor.

Example 21 Mouse Zalpha11 Ligand is Active in Mouse Bone Marrow Assay

A. Isolation of Non-Adherent Low Density Marrow Cells:

Fresh mouse femur aspirate (marrow) was obtained from 6-10 week old maleBalb/C or C57BU6 mice. The marrow was then washed with RPMI+10% FBS(JRH, Lenexa KS; Hyclone, Logan Utah) and suspended in RPMI+10% FBS as awhole marrow cell suspension. The whole marrow cell suspension was thensubjected to a density gradient (Nycoprep, 1.077, Animal; Gibco BRL) toenrich for low density, mostly mononuclear, cells as follows: The wholemarrow cell suspension (About 8 ml) was carefully pipeted on top ofabout 5 ml Nycoprep gradient solution in a 15 ml conical tube, and thencentrifuged at 600×g for 20 minutes. The interface layer, containing thelow density mononuclear cells, was then removed, washed with excessRPMI+10% FBS, and pelleted by centrifugation at 400×g for 5-10 minutes.This pellet was resuspended in RPMI +10% FBS and plated in a T-75 flaskat approximately 106 cells/ml, and incubated at 37° C. 5% CO₂ forapproximately 2 hours. The resulting cells in suspension wereNon-Adherent Low Density (NA LD) Marrow Cells.

B. 96-Well Assay

NA LD Mouse Marrow Cells were plated at 25,000 to 45,000 cells/well in96 well tissue culture plates in RPMI +10% FBS +1 ng/mL mouse Stem CellFactor (mSCF) (R&D Systems, Minneapolis, Minn.), plus 5% conditionedmedium from one of the following: (1) BHK 570 cells expressing mousezalpha11 Ligand (Example 18), (2) BHK 570 cells expressing humanzalpha11 Ligand (Example 7), or (3) control BHK 570 cells containingvector and not expressing either Ligand. These cells were then subjectedto a variety of cytokine treatments to test for expansion ordifferentiation of hematopoietic cells from the marrow. To test, theplated NA LD mouse marrow cells were subjected to human Interleukin-15(hIL-15) (R&D Systems), or one of a panel of other cytokines (R&DSystems). Serial dilution of hIl-15, or the other cytokines, weretested, with 2-fold serial dilution from about 50 ng/ml down to about6025 ng/ml concentration. After 8 to 12 days the 96-well assays werescored for cell proliferation by Alamar blue assay as described inExample 5B.

C. Results from the 96-Well NA LD Mouse Marrow Assay

Conditioned media from the BHK cells expressing both mouse and humanzalpha11 Ligand acted in synergy with hIL-15 to promote the expansion ofa population of hematopoietic cells in the NA LD mouse marrow. Thisexpansion of hematopoietic cells was not shown with control BHKconditioned medium plus IL-15. The population hematopoietic cellsexpanded by the mouse zalpha11 Ligand with hIL-15, and thosehematopoietic cells expanded by the human zalpha11 Ligand with hIL-15,were further propagated in cell culture. These hematopoietic cells werestained with a Phycoerythrin labeled anti-Pan NK cell antibody(Pharmingen) and subjected to flow cytometry analysis, whichdemonstrated that the expanded cells stained positively for this naturalkiller (NK) cell marker.

The same 96-well assay was run, using fresh human marrow cells boughtfrom Poietic Technologies, Gaithersburg, Md. Again, in conjunction withIL-15, the mouse and human zalpha11 Ligand expanded a hematopoietic cellpopulation that stained positively for the NK cell marker using theantibody disclosed above.

Example 22 Constructs for Generating Human Zalpha11 Ligand TransgenicMice

A. Construct for Expressing Human Zalpha11 Ligand from theLiver-Specific MT-1 Promoter

Oligonucleotides were designed to generate a PCR fragment containing aconsensus Kozak sequence and the human zalpha11 Ligand coding region.These oligonucleotides were designed with an FseI site at the 5′ end andan AscI site at the 3′ end to facilitate cloning into (a) pMT12-8, ourstandard transgenic vector, or (b) pKFO51, a lymphoid-specifictransgenic vector (Example 22B).

PCR reactions were carried out with 200 ng human zalpha11 Ligandtemplate (Example 7) and oligonucleotides ZC22,143 (SEQ ID NO:61) andZC22,144 (SEQ ID NO:62). PCR reaction conditions were as follows: 95° C.for 5 minutes, wherein Advantage™ cDNA polymerase (Clontech) was added;15 cycles of 95° C. for 60 seconds, 60° C. for 60 seconds, and 72° C.for 90 seconds; and 72° C. for 7 minutes PCR products were separated byagarose gel electrophoresis and purified using a QiaQuick™(Qiagen) gelextraction kit. The isolated, 488 bp, DNA fragment was digested withFseI and AscI (Boerhinger-Mannheim), ethanol precipitated and ligatedinto pMT12-8 previously digested with FseI and AscI. The pMT12-8plasmid, designed for expressing a gene of interest in liver and othertissues in transgenic mice, contains an expression cassette flanked by10 kb of MT-1 5′ DNA and 7 kb of MT-1 3′ DNA. The expression cassettecomprises the MT-1 promoter, the rat insulin II intron, a polylinker forthe insertion of the desired clone, and the human growth hormone (hGH)poly A sequence.

About one microliter of each ligation reaction was electroporated intoDH10B ElectroMax™ competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies werepicked and grown in LB media containing 100 μg/ml ampicillin. MiniprepDNA was prepared from the picked clones and screened for the humanzalpha11 Ligand insert by restriction digestion with EcoRI alone, orFsel and AscI combined, and subsequent agarose gel electrophoresis.Maxipreps of the correct pMT-human zalpha11 Ligand were performed. ASalI fragment containing with 5′ and 3′ flanking sequences, the MT-1promoter, the rat insulin II intron, human zalpha11 Ligand cDNA and thehGH poly A sequence was prepared to be used for microinjection intofertilized murine oocytes. Microinjection and production of transgenicmice were produced as described in Hogan, B. et al. Manipulating theMouse Embryo, 2^(nd) ed., Cold Spring Harbor Laboratory Press, N.Y.,1994.

B. Construct for Expressing Human Zalpha11 Ligand from theLymphoid-Specific EμLCK Promoter

Oligonucleotides were designed to generate a PCR fragment containing aconsensus Kozak sequence and the human zalpha11 Ligand coding region.These oligonucleotides were designed with an FseI site at the 5′ end andan AscI site at the 3′ end to facilitate cloning into pKFO51, alymphoid-specific transgenic vector.

PCR reactions were carried out with 200 ng human zalpha11 Ligandtemplate (Example 7) and oligonucleotides ZC22,143 (SEQ ID NO:61) andZC22,144 (SEQ ID NO:62). A PCR reaction was performed using Advantage™cDNA polymerase (Clontech) under the following conditions: 95° C. for 5minutes; 15 cycles of 95° C. for 60 seconds, 60° C. for 60 seconds, and72° C. for 90 seconds; and 72° C. for 7 minutes. PCR products purifiedas described above. The isolated, 488 bp, DNA fragment was digested withFseI and AscI (Boerhinger-Mannheim), ethanol precipitated and ligatedinto pKFO51 previously digested with FseI and AscI. The pKFO51transgenic vector is derived from p1026× (Iritani, B. M., et al., EMBOJ. 16:7019-31, 1997) and contains the T cell-specific lck proximalpromoter, the B/T cell-specific immunoglobulin μ heavy chain enhancer, apolylinker for the insertion of the desired clone, and a mutated hGHgene that encodes an inactive growth hormone protein (providing 3′introns and a polyadenylation signal).

About one microliter of each ligation reaction was electroporated,plated, clones picked and screened for the human zalpha11 Ligand insertby restriction digestion as described above. A correct clone ofpKFO51-zalpha11 Ligand was verified by sequencing, and a maxiprep ofthis clone was performed. A NotI fragment, containing the lck proximalpromoter and immunoglobulin μ enhancer (EμLCK), zalpha11 Ligand cDNA,and the mutated hGH gene was prepared to be used for microinjection intofertilized murine oocytes.

Example 23 Mouse Zalpha11 Ligand Tissue Distribution

Murine Multiple Tissue Northern Blots (Mouse, Mouse Embryo, Clontech;MB1010, MB1012 Origene) were probed to determine the tissue distributionof murine zalpha11 Ligand expression. An approximately 484 bp PCRderived probe was amplified using the plasmid M11L/pZP7 (Example 16) asa template and oligonucleotide ZC22283 (SEQ ID NO:57) and ZC22284 (SEQID NO:58) as primers. The PCR amplification was carried out as follows:1 cycle at 94° C. for 1.0 minutes; 35 cycles of 94° C. for 30 seconds,50° C. for 30 seconds, and 72° C. for 30 seconds; followed by 1 cycle at72° C. for 10 minutes. The PCR products were visualized by agarose gelelectrophoresis and the approximately 484 bp PCR product was purifiedusing a Gel Extraction Kit (Qiagen) according to manufacturer'sinstructions. The probe was radioactively labeled using the REDIPRIME™labeling kit (Amersham) according to the manufacturer's instructions.The probe was purified using a NUCTRAP™ push column (Stratagene).EXPRESSHYB™ (Clontech) solution was used for prehybridization and as ahybridizing solution for the Northern blots. Hybridization took placeovernight at 65° C. using 10⁶ cpm/ml of labeled probe. The blots werethen washed three times in 2×SSC and 0.1% SDS at room temperature,followed by 2 washes in 0.1×SSC and 0.1% SDS at 55° C. Two transcriptsof approximately 1.2 and 3.5 kb were seen in testis. The uppertranscript only was seen in thymus.

A murine RNA Master Dot Blot (Clontech) that contained RNAs from varioustissues that were normalized to 8 housekeeping genes was also probed andhybridized as described above. Expression was seen in testis.

Example 24 Purification of Untagged Human and Murine Zalpha11 Ligandfrom BHK 570

Unless otherwise stated, all operations were carried out at 4° C. Thefollowing procedure was used for purifying human and murine zalpha11Ligand from conditioned media from BHK 570 cells transfected with aconstruct expressing either the human zalpha11 Ligand (Example 25) orthe mouse zalpha11 Ligand (M11L/pZP9) (Example 18). The conditionedmedia was concentrated by standard techniques. Concentrated conditionedmedia (CM) was sterile filtered through 0.45 and 0.22 micron filters.The media was then diluted to low ionic strength (≦2 mS) in 0.01 M HEPES(JRH Biosciences, Lenexa, Kans.) at pH 7.0. The low ionic strengthdiluted CM was then loaded onto a 10×66 mm (6 ml) Poros HS 50 column(PerSeptive BioSystems, Framingham, Mass.) overnight at 4 ml/min using aBioCAD SPRINT (Perceptive BioSystems). The column was washed for 10-20column volumes (CV) with 0.01 M HEPES pH7.0. The bound proteins werethen step eluted with 1 M NaCl (Mallinckrodt, Paris, Ky.) in 0.01 MHEPES pH 7.0 at 5 ml/min; two ml fractions were collected over theentire chromatography and absorbence at 280 and 215 nM were monitored.Peak absorbence fractions were analyzed by bioassay and by SDS-PAGESilver (Geno Technology, St. Louis, Mo.) and Coomassie (Sigma, St.Louis, Mo.) staining. Peak fractions were pooled, sterile filtered anddiluted to ≦19 mS with Phosphate buffered saline (PBS, Gibco BRL)at pH7.2.

The diluted sample was then loaded at 2 ml/min using a BioCad SPRINT,onto either a 0.8 ml Poros AL column that had zalpha11CFLAG solublereceptor (Example 10B) or zalpha11-Fc4 fusion soluble receptor (Example10C) immobilized on the resin (see, below). The column was then washedwith at least 20 CV of PBS at 10 ml/min. The column was then rapidlyeluted with a 600 μl injection of 0.1 M glycine (Aminoacetic Acid;Glycocol, Spectrum, Gardena, Calif.) pH 2.5 at a flow rate of 10 ml/minwith PBS on a BioCAD 700E. The 1 ml fractions were collected for 6seconds each and immediately pH neutralized with 55 μl of 2 M TRIS (Tris(Hydroxymethyl) Aminomethane, EM Science, Gibbstown, N.J.) pH 8.8. Theabsorbence at 280 and 215 nM were monitored over the entirechromatography.

The peak fractions were analyzed by bioassay and by SDS-PAGE Silver(Geno Technology) and Coomassie (Sigma) staining. Two bands,approximately 24 kD and 18 kD, were seen on both Silver and Coomassiegels for mouse zalpha11 Ligand. A single band, at approximately 18 kD,was seen on both Silver and Coomassie gels for human zalpha11 Ligand.

Immobilization of Human Zalpha11 Soluble Receptor Polypeptides on POROSAL Media

Poros AL columns having immobilized zalpha11CFLAG soluble receptor(Example 10B) or zalpha11-Fc4 fusion soluble receptor (Example 10C) wereprepared. Approximately 3 mg of zalpha11CFLAG soluble receptor andapproximately 10 mg of zalpha11-Fc4 fusion soluble receptor were used.All operations were carried out at room temperature on a BioCAD 700E. A4.5×50 mm column with the POROS AL media was flow packed in 2 M NaClaccording to manufactures specifications. The column was thenequilibrated in 1.1 M Na₂SO₄/50 mM NaPhosphate pH 7.2. The receptor wasconcentrated to 4 mg/ml using a Millipore 30 MWKO spin concentrator thendiluted 1:1 in 1.1 M Na₂SO₄/50 mM NaPhosphate pH 7.2. The column wasflowed at 2 ml/min in 1.1 M Na₂SO₄/50 mM NaPhosphate pH 7.2 and 100 μlinjections of the diluted ligand were made ever 9 CVs until a steadystate of saturation, or break through, was reached. A 62 CV gradient wasthen run from 1.1 M Na₂SO₄/50 mM NaPhosphate pH 7.2., to 550 mMNa₂SO₄/50 mM NaPhosphate pH 7.2/5 mg/ml Sodium Cyanoborohydride. Thecolumn was then held for about 2 hours to complete the immobilizationchemistry. The column was then equilibrated in 0.2 M TRIS pH 7.2/5 mg/mlSodium Cyanoborohydride and allowed to rest for about 1 hour to cap thecolumn. Finally the column was equilibrated in PBS/0.02% Sodium Azide,and stored at 4° C. until needed. Prior to use, the column waspre-eluted with 0.1 M glycine to ensure that non-specific proteins wereremoved and that the column was not leaching the immobilized humanzalpha11 soluble receptor.

Example 25 Expression of Human Zalpha11 Ligand in Mammalian Cells

A. Construction of Expression Vector PZMP11/Zalpha11Lig

An expression plasmid containing all or part of a polynucleotideencoding human zalpha11 Ligand was constructed via homologousrecombination. A fragment of human zalpha11 Ligand cDNA (SEQ ID NO:63)was isolated using PCR.

Two primers were used in the production of the human zalpha11 Ligandfragment in a PCR reaction: (1) Primer ZC22,052 (SEQ ID NO:64),containing 40 bp of the vector flanking sequence and 17 bp correspondingto the amino terminus of the human zalpha11 Ligand, and (2) primerZC22,053 (SEQ ID NO:65), containing 40 bp of the 3′ end corresponding tothe flanking vector sequence and 17 bp corresponding to the carboxylterminus of the human zalpha11 Ligand. The PCR Reaction conditions wereas follows: 1 cycle at 94° C. for 2.0 minutes; 25 cycles of 94° C. for30 seconds, 60° C, for 30 seconds, and 72° C. for 30 seconds; followedby 1 cycle at 72° C. for 5 minutes; 4 C soak. Ten μl of the 100 μl PCRreaction was run on a 0.8% LMP agarose gel (Seaplaque GTG) with 1×TBEbuffer for analysis, and the expected approximately 560 bp fragmentseen. The remaining 90 μl of PCR reaction was precipitated with theaddition of 5 μl 1 M NaCl and 250 μl of absolute ethanol to be used forrecombining onto the recipient vector pZMP11 as described below. Therecipient plasmid pZMP 11 was previously cut with SmaI.

Plasmid pZMP11 was derived from the plasmid pCZR199 (described herein,e.g., Example 8). The plasmid pCZR199 is a mammalian expression vectorcontaining an expression cassette having the CMV immediate earlypromoter, a consensus intron from the variable region of mouseimmunoglobulin heavy chain locus, multiple restriction sites forinsertion of coding sequences, a stop codon and a human growth hormoneterminator. The plasmid also has an E. coli origin of replication, amammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a DHFR gene and the SV40 terminator.The vector pZMP11 was constructed from pCZR199 and includes thereplacement of the metallothionein promoter with the CMV immediate earlypromoter, and Kozac sequences at the 5′ end of the open reading frame.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl of a mixture containing approximately 1 μg of thehuman zalpha11 Ligand insert, and 100 ng of SmaI digested pZMP11 vector,and transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures were electropulsed at 0.75 kV (5 kV/cm), infinite ohms, 25 μF.To each cuvette was added 600 μl of 1.2 M sorbitol and the yeast wasthen plated in two 300 μl aliquots onto two URA-D plates and incubatedat 30° C.

After about 48 hours, the Ura+ yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 1 ml of lysis buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) wasdone with 0.5-2 ml yeast DNA prep and 40 μl of DH10B cells. The cellswere electropulsed at 2.0 kV, 25 mF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) was plated in 250 μl aliquots on four LB AMPplates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/LAmpicillin).

Individual clones harboring the correct expression construct for humanzalpha11 Ligand were identified by restriction digest to verify thepresence of the insert and to confirm that the various DNA sequenceshave been joined correctly to one another. The insert of positive cloneswere subjected to sequence analysis. Larger scale plasmid DNA wasisolated using the Qiagen Maxi kit (Qiagen) according to manufacturer'sinstruction

B. Mammalian Expression of Human Zalpha11 Ligand

BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm tissue culturedishes and allowed to grow to approximately 50 to 70% confluenceovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone,Logan, Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mMsodium pyruvate (Gibco BRL). The cells were then transfected with theplasmid PZMP11/zalpha11Lig (Example 25A), using Lipofectamine™ (GibcoBRL), in serum free (SF) media formulation (DMEM, 10 mg/ml transferrin,5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).Zalpha11-Fc4/pZMP6 (Example 8B) was diluted into 15 ml tubes to a totalfinal volume of 640 μl with SF media. 35 μl of Lipofectamine™ (GibcoBRL) was mixed with 605 μl of SF medium. The Lipofectamine™ mix wasadded to the DNA mix and allowed to incubate approximately 30 minutes atroom temperature. Five milliliters of SF media was added to theDNA:Lipofectamine™ mixture. The cells were rinsed once with 5 ml of SFmedia, aspirated, and the DNA:Lipofectamine™ mixture was added. Thecells were incubated at 37° C. for five hours, then 6.4 ml of DMEM/10%FBS, 1% PSN media was added to each plate. The plates were incubated at37° C. overnight and the DNA:Lipofectamine™ mixture was replaced withfresh 5% FBS/DMEM media the next day. On day 5 post-transfection, thecells were split into T-162 flask in selection medium (DMEM/5% FBS, 1%L-GLU, 1% NaPyr). Approximately 10 days post-transfection, two 150 mmculture dishes of methotrexate resistant colonies from each transfectionwere trypsinized and the cells are pooled and plated into a T-162 flaskand transferred to large scale culture. Conditioned media from largescale culture was used to purify human zalpha11 Ligand polypeptide asdescribed in Example 24.

Example 26 Construct for Generating Mouse Zalpha11 Ligand TransgenicMice

A. Construct for Expressing Mouse Zalpha11 Ligand from theLymphoid-Specific EμLCK Promoter

Oligonucleotides were designed to generate a PCR fragment containing aconsensus Kozak sequence and the mouse zalpha11 Ligand coding region.These oligonucleotides were designed with an FseI site at the 5′ end andan AscI site at the 3′ end to facilitate cloning into: (a) pKFO51, alymphoid-specific transgenic vector, or (b) pTG12-8, our standardtransgenic vector.

PCR reactions were carried out with 200 ng mouse zalpha11 Ligandtemplate (SEQ ID NO:55; Example 16) and oligonucleotides ZC23,115 (SEQID NO:66) and ZC23,116 (SEQ ID NO:67). A PCR reaction was performedusing Advantage™ cDNA polymerase (Clontech) under the PCR conditionsdescribed in Example 22B. PCR product was isolated as described inExample 22B. The isolated, 440 bp DNA fragment was digested and ligatedinto pKFO51 previously digested with FseI and AscI, as described inExample 22B.

About one microliter of each ligation reaction was electroporated,plated, clones picked and screened for the human zalpha11 Ligand insertby restriction digestion as described in Example 22. A correctpKFO51-zalpha11 Ligand clone was verified by sequencing, and a maxiprepof this clone was performed. A NotI fragment, containing the lckproximal promoter, immunoglobulin μ enhancer, zalpha11 Ligand cDNA, andthe mutated hGH gene was prepared to be used for microinjection intofertilized murine oocytes.

B. Construct for Expressing Mouse Zalpha11 Ligand from theLiver-Specific MT-1 Promoter

This same mouse zalpha11 Ligand insert from Example 26A, was subclonedinto the pTG12-8 vector, as described in Example 22A. For thisconstruct, about 10 mg of the pKFO51-zalpha11 Ligand maxiprep DNA wasdigested with FseI and AscI combined, ethanol precipitated, and themouse zalpha11 Ligand fragment was purified as described in Example 22.This fragment was then ligated into pTG12-8 which had been previouslydigested with FseI and AscI, as described in Example 22A.Electroporation, screening of clones and a maxiprep was performed asdescribed in Example 22. A SalI fragment containing 5′ and 3′ flankingsequences, the MT-1 promoter, the rat insulin II intron, mouse zalpha11Ligand cDNA and the hGH poly A sequence was prepared to be used formicroinjection into fertilized murine oocytes.

Example 27 Mouse Zalpha11-Ligand Polyclonal Antibodies

Polyclonal antibodies were prepared by immunizing 2 female New Zealandwhite rabbits with the purified recombinant protein muzalpha11L/MBP-6H(Example 32). The rabbits were each given an initial intraperitoneal(ip) injection of 200 mg of purified protein in Complete Freund'sAdjuvant followed by booster ip injections of 100 mg peptide inIncomplete Freund's Adjuvant every three weeks. Seven to ten days afterthe administration of the second booster injection (3 total injections),the animals were bled and the serum was collected. The animals were thenboosted and bled every three weeks.

The muzalpha11L/MBP-6H specific rabbit serum was pre-adsorbed ofanti-MBP antibodies using a CNBr-SEPHAROSE 4B protein column (PharmaciaLKB) that was prepared using 10 mg of purified recombinant maltosebinding protein (MBP) per gram of CNBr-SEPHAROSE. Recombinant MBP wasmade and purified on an amylose column in house, using methods wellknown in the art. The muzalpha11-ligand-specific polyclonal antibodieswere affinity purified from the rabbit serum using a CNBr-SEPHAROSE 4Bprotein column (Pharmacia LKB) that was prepared using 10 mg of thespecific antigen purified recombinant protein muzalpha11L/MBP-6H(Example 32) followed by 20× dialysis in PBS overnight.Muzalpha11-ligand-specific antibodies were characterized by ELISA using1 ug/ml of the purified recombinant proteins muzalpha11L/MBP-6H (Example32) or huzalpha11L-MBP/6H (Example 32) as antibody targets. The lowerlimit of detection (LLD) of the rabbit anti-muzalpha11L/MBP-6H affinitypurified antibody is 100 pg/ml on its specific purified recombinantantigen muzalpha11L/MBP-6H and 500 pg/ml on purified recombinanthuzalpha11L-MBP/6H.

Example 28 Construction of Mammalian Expression Vector and Large-ScaleHuman Zalpha11 Ligand Expression in CHO DG44 Cells

A mammalian expression vector for human zalpha11 Ligand (SEQ ID NO:1)designed to add a SalI site at the 5′ end and a PmeI site to the 3′ endof the cDNA, was constructed via amplification by PCR from a plasmidcontaining human zalpha11 Ligand (Example 7) with oligonucleotideprimers, ZC22,054 (SEQ ID NO:70) and ZC22,055 (SEQ ID NO:71). The PCRreaction conditions were as follows: 94° C. for 4 min.; 25 cycles of 94°C. for 45 sec., 50° C. for 45 seconds, and 72° C. for 3 min.; and 72° C.for 10 minutes. The PCR product was isolated as described herein, andcut with SalI and PmeI then ligated to plasmid pDC312 previously cut atthe appropriate restriction sites in the polylinker, using standardmethods described herein. The plasmid pDC312 is described in Morris, A.et al., “Expression Augmenting Sequence Element (EASE) isolated fromChinese Hamster Ovary Cells,” in Animal Cell Technology, Carrondo, M J Tet al (eds.) (1997) Kluwer Academic Publisers, The Netherlands, p.529-534.

The ligated vector was transfected into suspension-adapted CHO DG44 (inhouse Novo Nordisk, Denmark) cells by lipofection usingLipofectaminePlus™ reagent (Gibco/BRL) according to manufacturer'sinstructions. Transfectants were selected on PFCHO medium (JRH, Lenexa,Kans.) free of thymidine, and hypoxanthine, followed by selection on 200nM methotrexate (Sigma, St. Louis, Mo.). The methotrexate resistantcells were cloned by dilution and assayed for production of zalpha11Ligand by a BaF3 activity assay (Example 5B).

A productive clone was scaled up and grown in a 7 to 20 liter bioreactor(Applikon Bioreactors, Schiedam, The Netherlands) in PFCHO medium toproduce material for purification (Example 29).

Example 29 Large-Scale Purification of Untagged Human and MurineZalpha11 Ligand from BHK and CHO Mammalian Expression Cell Lines

A. CHO Expressed Human Zalpha11 Ligand

Unless otherwise stated, all operations were carried out at 4° C. Thefollowing procedure was used for purifying human zalpha11 Ligand from atleast 30 liters of CHO conditioned media (see, Example 28). Concentratedor non-concentrated conditioned media (CM) was sterile filtered through0.45 and 0.22 micron filters. The conditioned media was then bufferedwith 0.01 M MES (Fluka BioChemika, Switzerland)) and the pH adjusted to6.0, and then loaded onto a 50×100 mm (196 ml) Poros 50 HS column(strong cation exchanger from PerSeptive BioSystems, Framingham, Mass.)overnight at 4-10 ml/min. using a BioCAD SPRINT (Perceptive BioSystems).The column was washed for 10-20 column volumes (CV) with 0.01 MMES/0.130 M NaCl (Mallinckrodt, Paris, Ky.) pH 6.0. The bound proteinswere then eluted with a 0.130 M to 1 M NaCl 10 CV gradient in 0.01 M MESpH 6.0 at 30 ml/min.; 25 ml fractions were collected over the entirechromatography and absorbence at 280 and 215 nM were monitored. Peakabsorbence fractions were analyzed by SDS-PAGE Silver (Geno Technology,St. Louis , Mo.), Coomassie (Sigma, St. Louis, Mo.) staining and Westernimmunological blotting using antibodies against the human zalpha11Ligand (Example 33 and Example 34).

Peak fractions were pooled then concentrated in a stirred cellconcentrator on a YM10 membrane (Millipore/Amicon, Bedford, Mass.) to anominal volume (1-10 ml). The sample was then loaded on an appropriateSephacryl S-200 (Pharmacia, Uppsala, Sweden) high resolution sizeexclusion column (52-600 ml) equilibrated in PBS (Gibco BRL) at flowrates 1-2 ml/ml; 1-2 ml fractions were collected over the entirechromatography and absorbence at 280 and 215 nM were monitored. Peakfractions were analyzed by SDS-PAGE Silver (Geno Technology, St. Louis,Mo.), and Coomassie (Sigma, St. Louis, Mo.) staining.

The fractions of interest were pooled and concentrated with Millipore 5kD MWKO centrifugal spin concentrators to a minimal volume. The finalproduct was then analyzed by SDS PAGE Coomassie staining (Sigma, St.Louis, Mo.), Western Immunological blotting, N-terminal sequencing,Amino Acid Analysis, and BCA (Pierce, Rockford, Ill.) for protein purityand concentration.

B. BHK 570 Expressed Murine Zalpha11 Ligand

Unless otherwise stated, all operations were carried out at 4° C. Thefollowing procedure was used for purifying murine zalpha11 Ligand fromBHK conditioned media (Example 18). Concentrated or non-concentratedconditioned media (CM) was sterile filtered through 0.45 and 0.22 micronfilters. The media was then buffered with 0.01 M MES (Fluka BioChemika,Switzerland)) and the pH adjusted to 6.0. The CM was analyzed, loadedand eluted on an AS column as described in Example 29A.

Fractions of interest were pooled then concentrated in a stirred cellconcentrator as in Example 29A, to a volume of 20-30 ml. The pH wasadjusted to 7.0 then the sample was loaded onto either a 0.8 ml Poros ALcolumn that had about 3 mg of zalpha11CFLAG tagged soluble receptor(Example 10B) or one with about 10 mg of zalpha11-Fc4 fusion receptor(Example 10C) immobilized on the resin (see method below) at 1 ml/min ona BioCAD SPRINT. The column was then washed with at least 20 CV of 0.3 MNaCl/PBS (Gibco BRL)/0.01 M MES at 10 ml/min. The column was then rapideluted with a 600 μl injection of 0.1 M glycine (Aminoacetic Acid;Glycocol, Spectrum, Gardena, Calif.) pH 2.5 at a flow rate of 10 ml/minwith PBS on a BioCAD SPRINT. The 1 ml fractions were collected for 6seconds each and immediately pH neutralized with 55 μl of 2 M TRIS pH8.8 (Tris (Hydroxymethyl) Aminomethane, EM Science, Gibbstown, N.J.).The absorbence at 280 and 215 nM were monitored over the entirechromatography. The peak fractions were analyzed SDS-PAGE Silver (GenoTechnology, St. Louis , Mo.), Coomasise (Sigma, St. Louis, Mo.) stainingand Western Immunological blotting as above.

Peak fractions were pooled then concentrated in a stirred cellconcentrator as in Example 29A to a minimal volume (1-10 ml). The samplewas then loaded, equilibrated and analyzed as in Example 29A, on anappropriate Sephacryl S-200 (Pharmacia) high resolution size exclusioncolumn. Peak fractions were analyzed by SDS-PAGE Silver (GenoTechnology, St. Louis , Mo.), and Coomasise (Sigma, St. Louis, Mo.)staining. The fractions of interest were pooled and concentrated andanalyzed as in Example 29A.

C. Protein Immobilization on POROS AL Media

All operations were carried out at room temperature on a BioCAD 700E.Flow packed a 4.5×50 mm column with the POROS AL media in 2 M NaClaccording to manufactures specifications. The column was thenequilibrated in 1.1 M Na₂SO₄ and 50 mM NaPhosphate pH 7.2. The receptorwas concentrated to 4 mg/ml using a Millipore 30 kD MWKO centrifugalspin concentrator then diluted 1:1 in 1.1 M Na₂SO₄ and 50 mM NaPhosphatepH 7.2. The column was flowed at 2 ml/min in 1.1 M Na₂SO₄ and 50 mMNaPhosphate pH 7.2 and 100 μl injections of the diluted ligand were madeever 9 CVs until a steady state of saturation or break through wasreached. A 62 CV gradient was then ran from 1.1 M Na₂SO₄ and 50 mMNaPhosphate pH 7.2 to 550 mM Na₂SO₄ and 50 mM NaPhosphate pH 7.2 with 5mg/ml Sodium Cyanoborohydride. Column was held for 2 hr. to complete theimmobilization chemistry. The column was then equilibrated in 0.2 M TRISpH 7.2 with 5 mg/ml Sodium Cyanoborohydride and allowed to rest for 1hr. Finally the column was equilibrated in PBS with 0.02% Sodium Azide,then stored at 4° C. until needed. Prior to use, the column waspre-eluted with 0.1 M glycine to ensure that non-specific proteins wereremoved and the column is not leaching the immobilized receptor.

Example 30 Expression Vector Construction, Expression and PurificationOf Untagged Human And Murine Zalpha11 Ligand From Baculovirus

A. Construct for Expressing Human Zalpha11 Ligand in Baculovirus

An expression vector, pzalpha11L, was prepared to express Human zalpha11Ligand polypeptides in insect cells. A 517 bp fragment containingsequence for Human zalpha11 Ligand and encoded BamH1 and XhoIrestriction sites on the 5′ and 3′ ends respectively, was generated byPCR amplification from a plasmid containing human zalpha11 Ligand cDNA(Example 7) using primers ZC23,444 (SEQ ID NO:74) and ZC23,445 (SEQ IDNO:75). The PCR reaction conditions were as follows: 1 cycle of 94 C for4 minutes, followed by 25 cycles of 94° C. for 45 seconds, 50° C. for 45seconds, and 72° C. for 2 minutes; 1 cycle at 72° C. for 10 min;followed by a 4° C. soak. The fragment was visualized by gelelectrophoresis (1% SeaPlaque/1% NuSieve). The band was excised, dilutedto 0.5% agarose with 2 mM MgCl₂, melted at 65° C., digested with BamH1and XhoI (Boerhinger Mannheim), and ligated into an BamH1/XhoI digestedbaculovirus expression vector, pZBV3L. The pZBV3L vector is amodification of the pFastBac1™ (Life Technologies) expression vector,where the polyhedron promoter has been removed and replaced with thelate activating Basic Protein Promoter. About 14 nanograms of therestriction digested zalpha1 Ligand insert and about 40 ng of thecorresponding vector were ligated overnight at 16° C.

The ligation mix was diluted 3 fold in TE (10 mM Tris-HCl, pH 7.5 and 1mM EDTA) and about 4 fmol of the diluted ligation mix was transformedinto DH5α Library Efficiency competent cells (Life Technologies)according to manufacturer's direction by heat shock for 45 seconds in a42° C. waterbath. The transformed DNA and cells were diluted in 450 μlof SOC media (2% Bacto™ Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1MNaCl, 1.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) and platedonto LB plates containing 100 μg/ml ampicillin. Clones were analyzed byrestriction digests and 1 μl of the positive clone was transformed into20 μl DH10Bac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg,Md.) according to manufacturer's instruction, by heat shock as describedabove. The transformed cells were then diluted in 980 μl SOC media (2%Bacto™ Tryptone, 0.5% Bacto™ Yeast Extract, 10 ml 1 M NaCl, 1.5 mM KCl,10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) out grown in shakingincubator at 37° C. for four hours and plated onto Luria Agar platescontaining 50 μg/ml kanamycin, 7 μg/ml gentamicin (Life Technologies),10 μg/ml tetracycline, IPTG (Pharmacia Biotech) and Bluo-Gal (LifeTechnologies). The plated cells were incubated for 48 hours at 37° C. Acolor selection was used to identify those cells having Human zalpha11Ligand encoding donor insert that had incorporated into the plasmid(referred to as a “bacmid”). Those colonies, which were white in color,were picked for analysis. Human zalpha11 Ligand Bacmid DNA was isolatedfrom positive colonies using the QiaVac Miniprep8 system (Qiagen)according the manufacturer's directions. Clones were screened for thecorrect insert by amplifying DNA using primers to the transposableelement in the bacmid via PCR using primers ZC447 (SEQ ID NO:76) andZC976 (SEQ ID NO:77). The PCR reaction conditions were as follows: 35cycles of 94° C. for 45 seconds, 50° C. for 45 seconds, and 72° C. for 5minutes; 1 cycle at 72° C. for 10 min.; followed by 4 C soak. The PCRproduct was run on a 1% agarose gel to check the insert size. Thoseclones having the correct insert were used to transfect Spodopterafrugiperda (Sf9) cells.

B. Expression and Generation of Material for Purification of HumanZalpha11 Ligand from Baculovirus

Sf9 cells were seeded at 5×10⁶ cells per 35 mm plate and allowed toattach for 1 hour at 27° C. Five microliters of human zalpha11 Ligandbacmid DNA (above) was diluted with 100 μl Sf-900 II SFM (LifeTechnologies). Six μl of CellFECTIN Reagent (Life Technologies) wasdiluted with 100 μl Sf-900 II SFM. The bacmid DNA and lipid solutionswere gently mixed and incubated 30-45 minutes at room temperature. Themedia from one plate of cells were aspirated, the cells were washed 1×with 2 ml fresh Sf-900 II SFM media. Eight hundred microliters of Sf-900II SFM was added to the lipid-DNA mixture. The wash media was aspiratedand the DNA-lipid mix added to the cells. The cells were incubated at27° C. for 4-5 hours. The DNA-lipid mix was aspirated and 2 ml of Sf-900II media was added to each plate. The plates were incubated at 27° C.,90% humidity, for 96 hours after which the virus was harvested.

For Primary Amplification Sf9 cells were grown in 50 ml Sf-900 II SFM ina 125 ml shake flask to an approximate density of 0.41-0.52×10⁵cells/ml. They were then infected with 150 μl of the virus stock fromabove and incubated at 27° C. for 3 days after which time the virus washarvested according to standard methods known in the art. A 500 μlsample submitted for activity in a BaF3 assay (Example 5) to show thatit was biologically active.

For Secondary Amplification Sf9 cells were grown in IL of Sf-900 II SFMin a 2800 ml shake flask to an approximate density of 0.5×10⁵ cells/ml.It was infected with 500 ul of the Primary viral stock from above andincubated at 27° C. for 4 days after which time the virus was harvestedaccording to standard methods known in the art. Virus was titered andgrown up large scale for purification of the baculovirus-produced humanzalpha11 Ligand (huzalpha11L-Bv), as described in Example 30 C andExample 30D, below.

C. Large-scaled Purification of Baculovirus Expressed Human/MurineZalpha11 Ligand

Unless otherwise stated, all operations were carried out at 4° C. Thefollowing procedure was used for purifying human zalpha11 Ligand(huzalpha11L-Bv) from BV conditioned media (Example 30B). Conditionedmedia (CM) was sterile filtered through 0.45 and 0.22 micron filters,then buffered with 0.01 M MES (Fluka BioChemika, Switzerland)) and thepH adjusted to 6.0 The CM was then loaded onto a POROS 50 HS column andrun, fractions collected, analyzed, as described in Example 29A.

The above peak fractions were pooled, concentrated run on a highresolution size exclusion column, and analyzed as described in Example29A.

The fractions of interest from the size exclusion column were pooled andconcentrated with 5 kD MWCO Millipore centrifugal spin concentrators toa minimal volume. The final product was then analyzed by SDS-PAGECoomassie (Sigma, St. Louis, Mo.), Western immunological blotting,N-terminal sequencing, Amino Acid Analysis, and CB (Pierce, Rockford,Ill.) for protein purity and concentration as described in Example 29A.Bulk protein was stored at −80° C.

D. Small Scale (<2 mg) Purification of Baculovirus-ExpressedHuman/Murine Zalpha11 Ligand

Unless other wise stated, all operations were carried out at 4° C. Thefollowing procedure was used for purifying <2 mg of human or murinezalpha11 Ligand from BV conditioned media. The CM was filtered, bufferedand pH adjusted as in Example 30C. The CM was then loaded, eluted andthe POROS 50 HS chromatography was analyzed as in Example 30C.

Fractions were pooled then concentrated via diafiltration in a stirredcell concentrator on a YM10 membrane (10 kD MWCO) (Millipore/Amicon,Bedford, Mass.) to a nominal volume (20-30 ml). The pH was adjusted to7.0 then the sample was loaded onto either a 0.8 ml Poros AL column thathad about 3 mg of zalpha11CFLAG soluble receptor (Example 10B) or onewith about 10 mg of zalpha11-Fc4 fusion soluble receptor (Example 10C)immobilized on the resin (see method in example 29C) at 1 ml/min on aBioCad SPRINT. The column was then washed with at least 20 CV of 0.3 MNaCl/PBS(Gibco BRL)/0.01 M MES at 10 ml/min. The column was then rapideluted with a 600 μl injection of 0.1 M glycine (Aminoacetic Acid;Glycocol, Spectrum, Gardena, Calif.) pH 2.5 at a flow rate of 10 ml/minwith PBS on a BioCAD SPRINT. The 1 ml fractions were collected for 6seconds each and immediately pH neutralized with 55 μl of 2 M TRIS (Tris(Hydroxymethyl) Aminomethane, EM Science, Gibbstown, N.J.) pH 8.8. Theabsorbence at 280 and 215 nM were monitored over the entirechromatography. Fractions were analyzed as above.

Peak fractions were pooled then concentrated via diafiltration in astirred cell concentrator on a YM10 membrane (10 kD MWCO)(Millipore/Amicon, Bedford, Mass.) to 1-2 ml. The sample was then loadedon an appropriate Sephacryl S-200 (Pharmacia, Uppsala, Sweden) highresolution size exclusion column equilibrated in PBS (Gibco BRL) at anoptimal flow rate; fractions were collected over the entirechromatography and absorbence at 280 and 215 nM were monitored.Fractions were analyzed as above.

The fractions of interest were pooled and concentrated with 5 Kd MWCOMillipore centrifugal spin concentrators to a nominal volume. The finalproduct was then analyzed by SDS-PAGE Coomassie (Sigma, St. Louis, Mo.),Western immunological blotting, N-terminal sequencing, Amino AcidAnalysis, and BCA (Pierce, Rockford, Ill.) for protein purity andconcentration. Bulk protein stored as described above.

E. Construct for Expressing Mouse Zalpha11 Ligand in Baculovirus:Pzalpha11lig.M

An expression vector, pzalpha11LM, was prepared to express Murinezalpha11 Ligand polypeptides in insect cells. A 413 bp fragmentcontaining sequence for Murine zalpha11 Ligand and encoded BspE1 andXbal restriction sites on the 5′ and 3′ ends, respectively, wasgenerated by PCR amplification from a plasmid containing murine zalpha11Ligand cDNA (Example 16) using primers ZC25,970 (SEQ ID NO:109) andZC25,969 (SEQ ID NO:110) utilizing the Expand High Fidelity PCR System(Boerhinger Mannheim) as per manufacturer's instructions. The PCRconditions were as follows: 1 cycle of 94° C. for 2 minutes, followed by35 cycles of 94° C. for 15 seconds, 50° C. for 30 seconds, and 72° C.for 2 minutes; 1 cycle at 72° C. for 10 min; followed by a 4° C soak. Asmall portion of the PCR product was visualized by gel electrophoresis(1% NuSieve agarose). The remainder of the fragment was purified usingthe Qiagen PCR purification kit as per manufacturer's instructions andeluted into 30 μl of H₂O. The fragment was then digested with Bspe1 andXbal (Boerhinger Mannheim) restriction enzymes at 37 C for about 2 h,then run on an agarose gel as described above. The band was excised,purified and eluted using the Qiagen gel extraction kit as permanufacturers instructions. The purified fragment was ligated into anBspe1/Xbal digested baculovirus expression vector, pZBV37L. The pZBV37Lvector is a modification of the pFastBac1™ (Life Technologies)expression vector, where the polyhedron promoter has been removed andreplaced with the late activating Basic Protein Promoter followed by thesecretory signal sequence from Ecdysteroid UDP-Glucosyltransferase(EGT). About 5 μl of the restriction digested Murine zalpha11 Ligandinsert and about 100 ng of the corresponding cut vector were ligatedovernight at 16° C. in about 20 μl. Five μl of the ligation mix waselectroporated into 50 μl Life Technologies DH12S electrocompetentbacterial cells utilizing a 2 mm cuvette with 2 kV, 25 μF and 400 ohmssettings. The electroporated cells were rescued in 1 ml of SOC media (2%Bacto™ Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose (Gibco BRL)) outgrown at 37° C.for about 1 hr and plated onto LB plates containing 100 μg/mlampicillin. DNA from clones was isolated and analyzed by restrictiondigests to identify positive clones. About 5 ng DNA from a positiveclone was transformed into 20 μl DH10Bac Max Efficiency competent cells(GIBCO-BRL, Gaithersburg, Md.) according to manufacturer's instruction,by heat shock for 45 seconds in a 42° C. water bath. The transformedcells were then diluted and grown as described in example 30A. Bacmidscontaining the murine zalpha11 Ligand insert were identified andisolated as described in Example 30A. Clones were screened for thecorrect insert by amplifying DNA using primers to the transposableelement in the bacmid via PCR using primers ZC447 (SEQ ID NO:76) andZC976 (SEQ ID NO:77). The PCR reaction conditions were as follows: 35cycles of 94° C. for 45 seconds, 50° C. for 45 seconds, and 72° C. for 5minutes; 1 cycle at 72° C. for 10 min.; followed by 4° C. soak. The PCRproduct was run on a 1% agarose gel to check the insert size. Thosehaving the correct insert were used to transfect Spodoptera frugiperda(Sf9) cells.

F. Expression and Generation of Material for Purification of MouseZalpha11 from Baculovirus

Sf9 cells were seeded at I million cells per 35 mm plate and allowed toattach for 1 hour at 27° C. The murine zalpha11 Ligand bacmid DNA wastransfected as described in Example 30B and the virus was harvested.

For primary amplification, Sf9 cells were seeded as above and 500 μl of72 hr post transfection supernatant was added and cultures were allowedto proceed for 96 hr. after which time the virus was harvested accordingto standard methods.

For Secondary amplification, Sf9 cells were seeded as above and 200 μlof the Primary viral stock was added. Cultures were incubated at 27° C.for 72 hr., after which time the virus was harvested according tostandard methods.

For Tertiary amplification, 10 μl of Secondary Amplified virus stock wasplaced on SF9s at 500,000 cells per well in 50 ml of SF900II media in a250 ml vol. shake flask for 6 days and virus was harvested as above.Virus was titered and grown up large scale for purification of thebaculovirus-produced murine zalpha11 Ligand (muzalpha11L-Bv), asdescribed in Example 30C and Example 30D.

Presence of predicted molecular weight protein in the supernatant wasdetermined by western analysis using an anti-muzalpha11L/MBP-6Hpolyclonal antibody (Example 27). BaF3 based proliferation assayanalysis (Example 5) also showed that the secreted ligand was active.

Example 31 Expression of Human and Mouse Zalpha11 Ligand in E. coli

A. Construction of Human Zalpha11Ligand-MBP Fusion Expression VectorPTAP98/Zalpha11 Ligand

An expression plasmid containing a polynucleotide encoding part of thehuman zalpha11 Ligand fused N-terminally to maltose binding protein(MBP) was constructed via homologous recombination. A fragment of humanzalpha11 Ligand cDNA (SEQ ID NO:1) was isolated using PCR. Two primerswere used in the production of the human zalpha11 Ligand fragment in aPCR reaction: (1) Primer ZC22,128 (SEQ ID NO:78) , containing 40 bp ofthe vector flanking sequence and 26 bp corresponding to the aminoterminus of the human zalpha11 Ligand, and (2) primer ZC22,127 (SEQ IDNO:79), containing 40 bp of the 3′ end corresponding to the flankingvector sequence and 28 bp corresponding to the carboxyl terminus of thehuman zalpha11 Ligand. The PCR reaction conditions were as follows: 25cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 1minute; followed by 4° C. soak, run in duplicate. Two μl of the 100 μlPCR reaction were run on a 1.0% agarose gel with 1×TBE buffer foranalysis, showing the expected band of approximately 472 bp. Theremaining 90 μl of PCR reaction was combined with the second PCR tubeprecipitated with 400 μl of absolute ethanol to be used for recombininginto the Sma1 cut recipient vector pTAP98 to produce the constructencoding the MBP-zalpha11 Ligand fusion, as described below.

Plasmid pTAP98 was derived from the plasmids pRS316 and pMAL-c2. Theplasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P.and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E. coliexpression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP98 was constructed usingyeast homologous recombination. 100 ng of EcoR1 cut pMAL-c2 wasrecombined with 1 μg Pvul cut pRS316, 1 μg linker, and 1 μg Sca1/EcoR1cut pRS316. The linker consisted of oligos ZC19,372 (SEQ ID NO:80) (100pmol): ZC19,351 (SEQ ID NO:81) (1 pmol): ZC19,352 (SEQ ID NO:82) (1pmol), and ZC19,371 (SEQ ID NO:83) (100 pmol) combined in a PCRreaction. Conditions were as follows: 10 cycles of 94° C. for 30seconds, 50° C. for 30 seconds, and 72° C. for 30 seconds; followed by4° C. soak. PCR products were concentrated via 100% ethanolprecipitation.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl of a mixture containing approximately 1 μg of thehuman zalpha11 Ligand PCR product, and 100 ng of SmaI digested pTAP98vector, and transferred to a 0.2 cm electroporation cuvette. Theyeast/DNA mixture was electropulsed at 0.75 kV (5 kV/cm), infinite ohms,25 μF. To each cuvette was added 600 μl of 1.2 M sorbitol. The yeast wasthen plated in two 300 μl aliquots onto two—URA D plates and incubatedat 30° C.

After about 48 hours, the Ura+ yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 1 ml of lysis buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

Transformation of electrocompetent E. coli cells (MC1061, Casadaban et.al. J. Mol. Biol. 138, 179-207) was done with 1 μl yeast DNA prep and 40μl of MC1061 cells. The cells were electropulsed at 2.0 kV, 25 μF and400 ohms. Following electroporation, 0.6 ml SOC (2% Bacto™ Tryptone(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mMKCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) was plated in one aliquoton LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/LAmpicillin).

Individual clones harboring the correct expression construct for humanzalpha11 Ligand were identified by expression. Cells were grown inSuperbroth II (Becton Dickinson) with 100 μg/ml of ampicillin overnight.50 μl of the overnight culture was used to inoculate 2 ml of freshSuperbroth II+100μg/ml ampicillin. Cultures were grown at 37° C.,shaking for 2 hours. 1 ml of the culture was induced with 1 mM IPTG. 2-4hours later the 250 μl of each culture was mixed with 250 μl acid washedglass beads and 250 μl Thorner buffer with 5% βME and dye (8M urea, 100mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples were vortexedfor one minute and heated to 65° C. for 5-10 minutes. 20 μl were loadedper lane on a 4%-12% PAGE gel (NOVEX). Gels were run in 1×MES buffer.The positive clones were designated pTAP126 and subjected to sequenceanalysis. The polynucleotide sequence of MBP-human zalpha11 Ligandfusion within pTAP126 is shown in SEQ ID NO:84, and the correspondingpolypeptide in SEQ ID NO:85.

B. Bacterial Expression of Human Zalpha11 Ligand.

One microliter of sequencing DNA was used to transform strain W3110(ATCC). The cells were electropulsed at 2.0 kV, 25 μF and 400 ohms.Following electroporation, 0.6 ml SOC (2% Bacto™ Tryptone (Difco,Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) was plated in one aliquot on LBAMP plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/LAmpicillin).

Individual were expressed. Cells were grown in Superbroth II (BectonDickinson) with 100 μg/ml of ampicillin overnight. 50 μl of theovernight culture was used to inoculate 2 ml of fresh Superbroth II+100μg/ml ampicillin. Cultures were grown at 37° C., shaking for 2 hours. 1ml of the culture was induced with 1 mM IPTG. 2-4 hours later the 250 μlof each culture was mixed with 250 μl acid washed glass beads and 250 μlThorner buffer with 5% βME and dye(8M urea, 100 mM Tris pH7.0, 10%glycerol, 2mM EDTA, 5% SDS). Samples were vortexed for one minute andheated to 65° C. for 10 minutes. 20 μl were loaded per lane on a 4%-12%PAGE gel (NOVEX). Gels were run in 1XMES buffer. The positive cloneswere used to grow up for protein purification of the huzalpha11L/MBP-6Hfusion protein (Example 32, below).

C. Construction of Mouse Zalpha11 Ligand-MBP Fusion Expression VectorpTAP98/Mouse Zalpha11 Ligand

An expression plasmid containing a polynucleotide encoding part of themouse zalpha11 Ligand fused N-terminally to maltose binding protein(MBP) was constructed via homologous recombination, as described inExample 31A. A fragment of mouse zalpha11 Ligand cDNA (SEQ ID NO:55) wasisolated using PCR. Two primers were used in the production of mousezalpha11 Ligand fragment in a PCR reaction: (1) Primer ZC22,849 (SEQ IDNO:86), containing 40 bp of the vector flanking sequence and 24 bpcorresponding to the amino terminus of the mouse zalpha11 Ligand, and(2) primer ZC22,850 (SEQ ID NO:87), containing 40 bp of the 3′ endcorresponding to the flanking vector sequence and 21 bp corresponding tothe carboxyl terminus of the mouse zalpha11 Ligand. The PCR reactionconditions were as per above. The approximately 450 bp fragment wascloned into pTAP98 as described above. Clones were transformed,identified and grown up as described above. The positive clones weredesignated pTAP134 and subjected to sequence analysis. Thepolynucleotide sequence of MBP-mouse zalpha11 Ligand fusion withinpTAP134 is shown in SEQ ID NO:88, and the corresponding polypeptidesequence shown in SEQ ID NO:89. The positive clones were used to grow upin E. coli as described above for protein purification of themuzalpha11L/MBP-6H fusion protein (Example 32).

Example 32 Purification of Zalpha11-MBP Ligand or Zalpha11-MBP Receptor

Unless otherwise stated, all operations were carried out at 4° C. Thefollowing procedure was used for purifying zalpha11-MBP Ligand fusionsfor human zalpha11-MBP Ligand (huzalpha11L/MBP-6H) or murinezalpha11-MBP Ligand (muzalpha11L/MBP-6H) from E. coli. Human or mousezalpha11-MBP receptor fusions were carried out using the same method.Pre-spun frozen E. coli paste was thawed and diluted into 2 liters ofBuffer B (0.02 M TRIS (EM Science); 0.2 M NaCl (Mallincrodt); 0.01 M2-mercapto-ethanol (EM Science); pH 8.0; with 5 mg/l Pepstatin A(Boerhinger Mannheim); 5 mg/l Aprotinin (Boerhinger Mannheim); and 1mg/l PMSF (Fluka)) plus 1-2 ml of an anti-foaming reagent AF289 antifoam(Sigma). The mixture was processed in a pre-chilled French Press celldisrupter (Constant Systems LTD) with 20-30 kPSI.

The lysate was then centrifuged at 18,000×g for 45 minutes at 4° C.;retained the supernatant. A 200 ml slurry of Amylose resin (New EnglandBioLabs), pre-equilibrated in Buffer A (0.02 M TRIS (EM Science); 0.2 MNaCl (Mallincrodt); 0.01 M 2-mercapto-ethanol (EM Science); pH 8.0), wasadded to the lysate supernatant and incubated overnight in 21 rollerbottles to allow for maximum batch absorption of the MBP fusion protein.The resin was washed in batch column format for ≧5 column volumes withBuffer A, then batch eluted with Buffer C (Buffer A with 0.02 M Maltose(Sigma)). Crude fractions were collected and monitored by absorbence 280nm.

The eluted protein was analyzed by SDS NuPAGE (NOVEX) Coomassie (Sigma)staining. Sample and bulk protein were stored at −80° C.

Example 33 Human Zalpha11 Ligand Polyclonal Antibodies

Polyclonal antibodies to Human zalpha11 Ligand were prepared byimmunizing 2 female New Zealand white rabbits with the purifiedrecombinant protein huzalpha11L/MBP-6H (Example 32) or the purified CHOrecombinant protein huzalpha11L-CHO (Example 29). The rabbits were eachgiven an initial intraperitoneal (ip) injection of 200 mg of purifiedprotein in Complete Freund's Adjuvant followed by booster ip injectionsof 100 mg purified protein in Incomplete Freund's Adjuvant every threeweeks. Seven to ten days after the administration of the second boosterinjection (3 total injections), the animals were bled and the serum wascollected. The animals were then boosted and bled every three weeks.

The rabbit serum raised to huzalpha11L/MBP-6H was pre-adsorbed ofanti-MBP antibodies using a CNBr-SEPHAROSE 4B protein column (PharmaciaLKB) that was prepared using 10 mg of purified recombinant MBP per gramof CNBr-SEPHAROSE (Pharmacia). Recombinant MBP was made and purified onan amylose column in house, using methods well known in the art. Thehuzalpha11-ligand-specific polyclonal antibodies were affinity purifiedfrom the rabbit serum using a CNBr-SEPHAROSE 4B protein column(Pharmacia LKB) that was prepared using 10 mg of the specific antigenpurified recombinant protein huzalpha11L/MBP-6H or 10 mg of the purifiedCHO recombinant protein huzalpha11L-CHO per gram of CNBr-SEPHAROSE,followed by 20× dialysis in PBS overnight. Huzalpha11-ligand-specificantibodies were characterized by ELISA using 1 μg/ml of the purifiedrecombinant proteins huzalpha11L/MBP-6H (Example 32), human zalpha11Ligand (huzalpha11L-CHO) (Example 29), or muzalpha11L-MBP/6H (Example32) as antibody targets.

The lower limit of detection (LLD) of the rabbit anti-huzalpha11L/MBP-6Haffinity purified antibody was 10 ng/ml on its specific purifiedrecombinant antigen huzalpha11L/MBP-6H, 500 pg/ml on purifiedrecombinant huzalpha11L-CHO, and 100 pg/ml on purified recombinantmuzalpha11L/MBP-6H (Example 32). The LLD of the rabbitanti-huzalpha11L-CHO affinity purified antibody was 20 pg/ml on itsspecific purified recombinant antigen huzalpha11L-CHO, 500 pg/ml onpurified recombinant huzalpha11L/MBP-6H , and 50 ng/ml on purifiedrecombinant muzalpha11L/MBP-6H.

Example 34 Human Zalpha11 Ligand Anti-Peptide Antibodies

Polyclonal human zalpha11 Ligand anti-peptide antibodies were preparedby immunizing 2 female New Zealand white rabbits with the human zalpha11Ligand peptide, huzalpha11L-1 (SEQ ID NO:72) or huzalpha11L-3 (SEQ IDNO:73). The peptides were synthesized using an Applied Biosystems Model431A peptide synthesizer (Applied Biosystems, Inc., Foster City, Calif.)according to manufacturer's instructions. The peptides were thenconjugated to the carrier protein keyhole limpet hemocyanin (KLH) withmaleimide-activation. The rabbits were each given an initialintraperitoneal (ip) injection of 200 mg of peptide in Complete Freund'sAdjuvant followed by booster ip injections of 100 mg peptide inIncomplete Freund's Adjuvant every three weeks. Seven to ten days afterthe administration of the second booster injection (3 total injections),the animals were bled and the serum was collected. The animals were thenboosted and bled every three weeks.

The rabbit sera raised to the human zalpha11 Ligand peptides werecharacterized by an ELISA titer check using 1 μg/ml of the respectivepeptide used to make the antibody (SEQ ID NO:72 or SEQ ID NO:73) as anantibody target. The 2 rabbit seras to the huzalpha11L-1 peptide hadtiter to their specific peptide at a dilution of 1:5,000,000 (1:5E6).The 2 rabbit seras to the huzalpha11L-3 peptide had titer to theirspecific peptide at a dilution of 1:5E6.

Human zalpha11 Ligand peptide-specific polyclonal antibodies wereaffinity purified from the rabbit serum using CNBR-SEPHAROSE 4B proteincolumns (Pharmacia LKB) that were prepared using 10 mg of the respectivespecific peptide (SEQ. ID. NO:72 or SEQ. ID. NO:73) per gramCNBr-SEPHAROSE, followed by 20× dialysis in PBS overnight. Huzalpha11-ligand-specific antibodies were characterized by an ELISA titer checkusing 1 μg/ml of the appropriate purified peptide antigen or purifiedrecombinant full-length proteins as antibody targets.

The lower limit of detection (LLD) of the rabbit anti-huzalpha11L-1affinity purified antibody is 500 pg/ml on its specific peptide antigen(huzalpha11L-1; SEQ ID NO:72), 500 pg/ml on purified recombinanthuzalpha11L/MBP-6H (Example 32), and 500 pg/ml on purified CHOrecombinant huzalpha11L-CHO (Example 29). No cross-reactivity was seento the purified recombinant muzalpha11L/MBP-6H (Example 32). The LLD ofthe rabbit anti-huzalpha11L-3 affinity purified antibody is 50 pg/ml onits specific peptide antigen (huzalpha11L-1; SEQ ID NO:73), 50 pg/ml onpurified recombinant huzalpha11L/MBP-6H, 500 pg/ml on purified CHOrecombinant huzalpha11L-CHO (Example 29), and 100 pg/ml on purifiedBaculovirus recombinant huzalpha11L-Bv (Example 30). Cross-reactivitywas seen to the purified recombinant muzalpha11L/MBP-6H (Example 32)with an LLD of 5 ng/ml.

Example 35 Human Zalpha11 Receptor Monoclonal Antibodies

Zalpha11 receptor Monoclonal antibodies were prepared by immunizing 5male BalbC mice (Harlan Sprague Dawley, Indianapolis, Ind.) with thepurified recombinant soluble receptor protein, zalpha11CEE(huzalpha11-CEE-BHK) (Example 10A). The mice were each given an initialintraperitoneal (IP) injection of 20 mg of purified protein in CompleteFreund's Adjuvant (Pierce, Rockford, Ill.) followed by booster IPinjections of 10 mg purified protein in Incomplete Freund's Adjuvantevery two weeks. Seven to ten days after the administration of the thirdbooster injection, the animals were bled and the serum was collected.

The mouse sera samples raised to the huzalpha11-CEE-BHK werecharacterized by an ELISA titer check using purified recombinant CHOhuzalpha11-Fc protein (Example 10C) as an antibody target. One mouseserum sample had titer to the specific antibody target at a dilution of1:1,000,000 (1:1E6). Four mouse serum samples had titer to the specificantibody target at a dilution of 1:100,000 (1:1E5).

Splenocytes were harvested from the 4 high-titer mice and fused tomurine SP2/0 myeloma cells using PEG 1500 (Boerhinger Mannheim, UK) intwo separate fusion procedures using a 4:1 fusion ratio of splenocytesto myeloma cells (Antibodies: A Laboratory Manual, E. Harlow and D.Lane, Cold Spring Harbor Press). Following 10 days growth post-fusion,specific antibody-producing hybridomas were identified by ELISA usingpurified recombinant BHK human zalpha11-Fc4 protein (Example 10C) as anantibody target and by FACS using Baf3 cells expressing the huzalpha11sequence (Example 4, and Example 2) as an antibody target. The resulting4 hybridomas positive by both methods were cloned three times bylimiting dilution. The antibodies were designated: 249.28.2.1.2.2;247.10.2.15.4.6; 249.19.2.2.3.5; and 249.15.2.4.2.7.

Example 36 Zalpha11 Ligand Transgenic Mice

A. Generation of Transgenic Mice Expressing Human and Mouse Zalpha11Ligand

DNA fragments from transgenic vectors (Example 22 and Example 26)containing 5′ and 3′ flanking sequences of the respective promoter (MT-1liver-specific promoter (mouse zalpha11 Ligand (Example 26B) or lymphoidspecific LCK promoter (mouse and human zalpha11 Ligand (Examples 26A and22B), the rat insulin II intron, zalpha11 Ligand cDNA and the humangrowth hormone poly A sequence were prepared and used for microinjectioninto fertilized B6C3f1 (Taconic, Germantown, N.Y.) murine oocytes, usinga standard microinjection protocol. See, Hogan, B. et al., Manipulatingthe Mouse Embryo. A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1994.

Eight transgenic mice expressing human zalpha11 Ligand from thelymphoid-specific EμLCK promoter were identified among 44 pups. Four ofthese were pups that died and 4 grew to adulthood. Expression levelswere fairly low in these animals. Twenty transgenic mice expressingmouse zalpha11Ligand from the lymphoid-specific EμLCK promoter wereidentified among 77 pups. All 20 grew to adulthood. Expression levelswere fairly low in these animals. Three transgenic mice expressing mousezalpha11 Ligand from the liver-specific MT-1 promoter were identifiedamong 60 pups. Two of these pups died and 1 grew to adulthood.Expression levels were fairly low in these animals. Tissues wereprepared and histologically examined as describe below.

B. Microscopic Evaluation of Tissues from Transgenic Mice

Spleen, thymus, and mesenteric lymph nodes were collected and preparedfor histologic examination from transgenic animals expressing human andmouse zalpha11 Ligand (Example 36A). Other tissues which were routinelyharvested included the following: Liver, heart, lung, kidney, skin,mammary gland, pancreas, stomach, small and large intestine, brain,salivary gland, trachea, espohogus, adrenal, pituitary, reproductivetract, accessory male sex glands, skeletal muscle including peripheralnerve, and femur with bone marrow. The tissues were harvested from aneonatal pup which died unexpectedly, and several adult transgenic mice,as described below. Samples were fixed in 10% buffered formalin,routinely processed, embedded in paraffin, sectioned at 5 microns, andstained with hematoxylin and eosin. The slides were examined and scoredas to severity of tissue changes (0=none, 1=mild, 2=moderate, 3=severe)by a board certified veterinary pathologist blinded to treatment.

The pup and 2 female adult mice expressing the human zalpha11 Ligand,and 3 of the 6 male adult mice expressing the mouse zalpha11 Ligandshowed inflammatory infiltrates in many of the tissues examined. Theorgans affected varied somewhat from mouse to mouse. The inflammatoryinfiltrate was composed primarily of neutrophils and macrophages invarying numbers and proportions and was generally mild to moderatedegree in severity. Moreover, these animals showed changes in lymphoidorgans, including moderate to severe lymphopenia in the spleen andthymus (human and mouse zalpha11 Ligand transgenics); and severelymphopenia (human zalpha11 Ligand transgenics), or mild to severesuppurative to pyogranulomatous lymphadenitis (mouse zalpha11 Ligandtransgenics) in lymph nodes. In addition, increased extramedullaryhematopoiesis was evident in the spleens. These changes were notobserved in age-matched control mice.

C. Flow Cytometric Analysis of Tissues from Transgenic Mice OverExpressing Zalpha11 Ligand

Transgenic animals over expressing either human or mouse zalpha11 ligand(Example 36A) were sacrificed for flow cytometric analysis of peripheralblood, thymus, lymph node, bone marrow, and spleen.

Cell suspensions were made from spleen, thymus and lymph nodes byteasing the organ apart with forceps in ice cold culture media (500 mlRPMI 1640 Medium (JRH Biosciences. Lenexa, Kans.); 5 ml 100× L-glutamine(Gibco BRL. Grand Island, N.Y.); 5 ml 100× Na Pyruvate (Gibco BRL); 5 ml100× Penicillin, Streptomycin, Neomycin (PSN) (Gibco BRL) and thengently pressing the cells through a cell strainer (Falcon, VWR Seattle,Wash.). Peripheral blood (200 ml) was collected in heparinized tubes anddiluted to 10 mls with HBSS containing 10 U Heparin/ml. Erythrocyteswere removed from spleen and peripheral blood preparations by hypotoniclysis. Bone marrow cell suspensions were made by flushing marrow fromfemurs with ice cold culture media. Cells were counted and tested forviability using Trypan Blue (GIBCO BRL, Gaithersburg, Md.). Cells wereresuspended in ice cold staining media (HBSS, 1% fetal bovine serum,0.1% sodium azide) at a concentration of ten million per milliliter.Blocking of Fc receptor and non-specific binding of antibodies to thecells was achieved by adding 10% normal goat sera and Fc Block(Pharmingen, La Jolla, Calif.) to the cell suspension.

Cell suspensions were mixed with equal volumes of fluorochrome labeledmonoclonal antibodies (PharMingen), incubated on ice for 60 minutes andthen washed twice with ice cold wash buffer (PBS, 1% fetal bovine serum,0.1% sodium azide) prior to resuspending in 400 ml wash buffercontaining 1 mg/ml 7-AAD (Molecular Probes, Eugene, Oreg.) as aviability marker in some samples. Flow data was acquired on aFACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose,Calif.). Both acquisition and analysis were performed using CellQuestsoftware (BD Immunocytometry Systems).

The transgenic animals that expressed either the human or mouse zalpha11Ligand at the highest levels had dramatically altered cell populationsin all lymphoid organs analyzed. Changes seen included complete loss ofthymic cellularity, complete absence of CD45R positive B cells andincreased size and cellularity of spleens. Both spleen and bone marrowhad increased numbers of myeloid sized cells, which was accounted for byincreases in both monocytes and neutrophils. The pan NK cell marker(DX5) was increased in many populations. Moderate expressing foundershad less dramatic but still significant changes consistent with thephenotype seen in the high expressers. Mice with the lowest level ofexpression had neither a significant increase in myeloid cells nordecrease in B cells numbers. They did show significant changes inthymocyte populations with decreases in CD4+CD8+ double positive cellsand increases in both CD4 and CD8 single positive cells.

Example 37 Zalpha11 Ligand Purified Recombinant Human ProteinDose-Response Study in Normal Mice

A. Summary

Normal six week old female C57B/6 (Harlan Sprague Dawley, Indianapolis,Ind.). mice were treated by intraperitoneal injection once daily foreither four or eight days with one of four dose levels of purifiedrecombinant human zalpha11 Ligand (Example 24) at 0.1, 0.5, 5 or 50μg/mouse/day or with vehicle as a control. Body weights and bodytemperatures were monitored daily. On either day four or day nine, fourof the eight mice from each protein treatment group and five of the tenmice in the vehicle control group were sacrificed. Blood, bone marrowand tissues were harvested and analyzed. Potential perturbations inlymphoid tissues were examined, as well as general physiologic andtoxicological parameters.

There was no evidence of toxicity of human zalpha11 Ligand protein atany of the doses tested. Body weights and temperatures were unchanged.There were no apparent changes in clinical chemistry parameters.However, there were consistent findings relating to increasedpercentages of myeloid lineage cells in bone marrow, spleen andperipheral blood in mice treated with the highest dose of zalpha11Ligand compared to the vehicle control. There was a statisticallysignificant increase in myeloid lineage sized cells identified by flowcytometric analysis of spleen homogenate in the high-dose group. Thespleens of the two highest dose groups were statistically significantlylarger than the other groups. On histopathologic examination, however,only a marginal increase in extramedullary hematopoiesis was seen in thehighest dose group. There was a statistically significant increase inthe myeloid to erythroid ratio of the bone marrow in the highest dosegroup compared to the other groups. Finally, there were increases seenin peripheral blood both in total white blood cell counts and in thepercentage of monocytes in the same group.

B. Dosing Solution Preparation

Purified recombinant human zalpha11 Ligand (Example 24) was diluted intosterile phosphate buffered saline (GibcoBRL, Grand Island, N.Y.) atconcentrations to deliver 50, 5, 0.5 or 0.1 micrograms of protein in 0.1ml of PBS vehicle. The doses for the first four days were made on day 0and frozen in a frosty −20° C. freezer prior to use. The doses for daysfive through eight were made on day five and frozen as above. Aliquotsof the same PBS were similarly frozen for the vehicle treated controlgroup. On the day of administration the appropriate aliquots were thawedand 0.1 ml of solution was injected intraperitoneally into the mice eachday for either four or eight days.

C. Study Design

The mice were six weeks old at the start of the study. Each treatmentgroup consisted of eight mice, except for the vehicle control group thatincluded ten mice. One half of the mice in each treatment group weresacrificed after four days of treatment and the other half after eightdays.

Before treatment each day, each mouse was weighed and her bodytemperature recorded using the Portable Programmable Notebook System(BMDS, Inc, Maywood, N.J.), by scanning the mouse for identificationnumber and body temperature from transponders implanted subcutaneously(IPTT-100, BMDS, Maywood, N.J.).

At sacrifice, tissues harvested to assess white blood cell populationsby flow cytometric analysis included bone marrow, thymus and spleen.FACS analysis of the lymphoid organs and bone marrow was performed withthe FACSCalibur, (Becton Dickinson, Mansfield, Mass.). The tissuesharvested for histologic examination for signs of toxicity of theprotein included: spleen, thymus, liver, kidney, adrenal gland, heartand lungs. All tissues fixed for histology were kept at 4° C. overnightin 10% Normal Buffered Saline (NBF) (Surgipath, Richmond, Ill.). Thefollowing day the NBF was replaced with 70% ethanol and the tissuesreturned to 4° C. until processing for histology.

The tissues were processed and stained for Hematoxylin and Eosin inhouse, then sent to a contract pathologist for histopathologic analysis.Blood was collected for complete blood cell counts (CBC) and serumchemistry profiles. The CBC's were analyzed in-house with the Cell Dyn3500 Hematology Analyzer (Abbott Diagnostics Division, Abbott Park,Ill.) and manual differential white blood cell counts were analyzed atPhoenix Central Laboratory, (Everett, Wash.). The serum was kept frozenat −20° C. until submission to Phoenix Central Laboratory for completeserum chemistry panels. To assess myeloid:erythroid ratios, the bonemarrow from one femur was applied to CytoSpin slides (CYTOSPIN 3CYTOCENTRIFUGE and CYTO SLIDES, Shandon, Pittsburgh, Pa.) and sent toPhoenix Central Laboratories for analysis.

D. Study Results

There were no apparent clinical indications of physiologic effects or oftoxicity of human zalpha11 Ligand at doses of 50μg/day or lower. Bodyweights and temperatures remained normal for the duration of thetreatments. Serum chemistry parameters were in normal ranges. Red bloodcell and platelet counts appeared normal. In the mice receiving 50g/dayfor 8 days, manual differential white blood cell counts showed that thepercentage of monocytes was elevated in the peripheral blood, and anapparent increase in the total white blood cell counts. In bone marrowflushed from a femur, myeloid to erythroid ratios were increased in the50 μg dose group, and to a lesser degree the 5μg dose group from the8-day dose set. In a non-parametric multiple column comparison usingInStat (InStat MAC; GraphPad Software, Inc., San Diego, Calif.), thisdifference was statistically significant (p=0.0049). The differencebetween the highest dose group and vehicle was also significant,(p=0.0286). The increased white blood cells in peripheral blood and thesignificant increase in myeloid precursors in the marrow may thus berelated.

Histologic evaluation of the following tissues showed no apparentevidence of cytologic or structural changes, mitotic events or necrosis:thymus, liver, kidney, adrenal gland, duodenum, pancreas, jejunum,caecum, colon, mesenteric lymph nodes, uterus, ovary, salivary gland,heart, trachea, lung, and brain. There were no apparent differencesbetween the treatment groups in the weights of the thymus, kidney, liveror brain. Of all the tissues examined, only the spleen weights weresignificantly affected.

Each mouse spleen weight was normalized to her brain weight. In the 50μg/day treatment group compared to the vehicle, 0.1 μg and 0.5 μgtreatment groups, the average of the spleen weights was nearly 50%greater after four days of treatment and almost 100% greater after eightdays than the average spleen weights of the other three groups. In thefour-day set, the 5 μg/day group also tended to have larger spleens thanthe control and low dose groups. The difference in the spleen/brainweights with data from the four-day and the eight-day sets combined bytreatment group was statistically significant (p=0.0072) byKruskall-Wallace non-parametric ANOVA, multiple column comparison testusing the InStat program (GraphPad Software).

A marginal increase in extrameduallary hematopoiesis, especially in thered pulp was seen in spleens of mice from the highest dose group, evenin the mice treated for four days. Flow cytometric analysis of thespleens showed a significant increase in the proportion of myeloid sizecells in the highest dose group (p=0.01, Student's t test), representingincreases in both monocytes and neutrophils. This effect may be relatedto the increased peripheral blood mononuclear cell percentage, as wellas the apparent increase in myeloid precursors in the bone marrow,described above. Moreover, the transgenic mice derived from insertion ofthe human zalpha11 gene had increased extramedullary hematopoiesis intheir spleens compared to non-transgenic litter mates.

Several changes were observed in the 50 μg per day dose group comparedto the control group that implicate zalpha11 Ligand in production ordevelopment of cells of the myeloid lineage. Taken together, theobserved changes suggest that zalpha11 may be useful as a therapeuticprotein in such medical specialties as cancer and immunologic disordersdescribed herein.

Example 38 Preliminary Elimination and Tissue Distribution Study OfPurified Recombinant Human Zalpha11 Ligand Protein

A. Summary

In order to elucidate tissue distribution and elimination patterns ofthe purified rhzalpha11 Ligand, a preliminary pharmacokinetic study wasundertaken. Nine week old male C57B1/6 mice were given purifiedrecombinant human zalpha11 Ligand protein labeled with ¹¹¹Indium (¹¹¹In)(NEN, Boston, Mass.) by one of three routes. A single bolus injectionwas given to each mouse by either the intravenous (IV), intraperitoneal(IP), or subcutaneous route (SC). The mice injected by either thesubcutaneous or intraperitoneal route were sacrificed at either one orthree hours after injection. The mice injected intravenously weresacrificed after either ten minutes or one hour following injection.Blood, plasma and selected tissues were harvested at various timepointsand counted by a gamma counter to estimate the approximate half-life andtissue distribution of the exogenous labeled protein. The tissues thatwere harvested for counting as well as the intervals of sacrifice wereselected based on reports of the distribution of other cytokines labeledwith radionuclides.

At sacrifice, tissues harvested for counting of radioactivity includedthymus, spleen, kidney, a lobe of liver, a lobe of lung, and urinarybladder. In the group receiving the injection intraperitoneally, gut wasalso counted to assess incidence of injection into the gut, and in thesubcutaneously dosed mice, skin with underlying structures in the areaof injection was counted. The cpm for whole liver and lung werecalculated from a section that was counted and a percentage of the wholeorgan weight represented by the section.

After the end of the study the collected tissues, whole blood and plasmawere counted on the COBRA II AUTO-GAMMA gamma counter (PackardInstrument Company, Meriden, Conn.). An aliquot of the original labeleddosing solution was also counted at the end of the study with thetissues. This allowed calculation of percent total injectedradioactivity for each mouse and simultaneous correction of all countsfor radioactive decay. Approximations of remaining blood volume andorgan weights indicated that the majority of the counts administeredwere accounted for, and therefore the percentage of counts per tissuewere a reasonable representation of distribution of the counts followinglabeled zalpha11 Ligand administration by each route.

B. ¹¹¹Indium Labeling of Zalpha11 Ligand

Purified recombinant human zalpha11 Ligand (Example 29) was conjugatedwith a 10 fold molar excess of DTPA (Peirce, Rockford, Ill.) byincubating 30 minutes at room temperature in PBS. Unreacted DTPA andhydrolyzates were removed by buffer exchange on a Biomax-5k NMWL(Ultrafree-15, Millipore, Bedford, Mass.). The void volume protein peakwas concentrated to 5 mg/ml and an aliquot taken for testing in abioassay (anti-CD40 stimulation of murine B-cells (Example 44)). Uponconfirming that the DTPA-conjugate still had full bioactivity theconjugate was diluted to 0.5 mg/ml with 1M Na Acetate pH6.0. Two mCi of¹¹¹Indium was taken up in 0.5 ml 1M Na Acetate pH6.0 and mixed with theDTPA-human zalpha11 Ligand for 30 min. at room temperature.Unincorporated ¹¹¹Indium was removed during buffer exchange to PBS on aPD-10 column (Pharmacia, Piscataway, N.J.). The radio-labeled materialwas diluted with unlabeled human zalpha11 Ligand to give a specificactivity of 100 mCi/mg, sterile filtered and stored at 4° C. overnight.One hundred percent of the labeled protein was retained on a Biomax-5kNMWL membrane (Millipore). The labeled ¹¹¹In-human zalpha11 Ligand wasadministered to mice in the elimination and pharmacokinetic studies.Fifty μg human zalpha11 Ligand protein labeled with 5 μCi of labeledhuman zalpha11 Ligand in 0.1 ml of PBS vehicle was administered to eachanimal.

C. Results Of Preliminary Distribution Study

After one and three hours following administration by all three routes,the highest concentration of ¹¹¹In-human zalpha11 Ligand, was found inkidney and the second highest was in urine and urinary bladder, asevinced by these tissues having the highest cpm. The average countsrecovered from kidneys were from 3 to 8 times higher than the wholeliver counts, depending on the route of injection and the sacrificetimepoint. For example, the average kidney cpm at 60 minutes followingIV injection was 4.5 times greater than the average counts calculatedfor whole liver from the same group. In the group that was sacrificedten minutes after intravenous administration, the highest cpm was againin kidney, and the second highest accumulation was equivalent in liver,urinary bladder and urine.

D. Preliminary Pharmacokinetic Study

Blood and plasma collections were done at 10, 30 and 60 minutesfollowing injection by all three routes. Following injection by the IVroute, a separate set of mice had blood and plasma samples taken at two,five and ten minutes. Another set of mice who received their injectionsby either the IP or SC route had blood sampled at one, two and threehours. For the treatment groups see Table 6. The short collection timesbracket the reported half-life of IL-2 following intravenous injection.The reported T1/2 was in the range of 2.5 to 5.1 minutes. For referenceto in vivo administration to IL-2, see Donohue J H and Rosenberg S A JImmunol, 130:2203, 1983. The long timepoints were chosen to outline theanticipated elimination phase.

TABLE 6 Route of injection Bleed Times(min.) Sacrifice Time IntravenousGroup 1  2, 5, 10  10 min. Intravenous Group 2 10, 30, 60  60 min.Intraperitoneal Group 1 10, 30, 60  60 min. Intraperitoneal Group 2 60,120, 180 180 min. Subcutaneous Group 1 10, 30, 60  60 min. SubcutaneousGroup 2 60, 120, 180 180 min.

Un-labeled IL-2 has been shown to be eliminated from the serum with ahalf-life of approximately three minutes in mice after IV injection. Forreference see Donahue, J H and Rosenburg supra. Following IP and SCinjection of similar amounts of L-2, the duration of persistence of IL-2activity in serum was prolonged from 2 units/ml for less than 30 minutesfollowing IV injection to greater than 2 units/ml for 2 hours followingIP and 6 hours following SC injections. The principle route of clearanceof L-2 appears to be the kidney. Zalpha11 ligand has been shown to bestructurally similar to IL-2, as discussed herein. Preliminaryevaluation of the elimination of zalpha11 Ligand appears to beconsistent with the apparent clearance of IL-2 by the kidneys, based onthe accumulation of cpm predominantly in the kidneys, followed by theurinary bladder and urine in the present study.

Estimations were made of pharmacokinetic parameters based on noncompartmental analysis of the cpm data obtained from the plasma, usingthe PK analysis program WinNonLin, Version 1.1, (Scientific ConsultingInc., Cary, N.C.). Plasma half-lives of zalpha11 Ligand were estimatedusing the predicted terminal elimination rate constants for intravenous,subcutaneous, and intraperitoneal administration of a 50 μg dose. Thepharmacokinetic results were estimations due to limited data points inthe terminal elimination region of the plasma concentration vs. timeprofiles. Moreover, the fit of the terminal elimination phase for SC andIP dosing required use of data from timepoints during which absorptionof the ¹¹¹In-human zalpha11 Ligand was apparently still occurring.However, estimations of half-lives following intravenous, subcutaneous,and intraperitoneal dosing were 13.6 min., 18.8 min., and 34.3 min.,respectively. Since a dosing range was not evaluated it was not apparentwhether saturable or active elimination (Michaelis Menten kinetics) wasoccurring. Therefore, these half-life calculations are estimations.

Estimates of the bioavailability of the labeled protein were made basedon the area under the curve (AUC) following subcutaneous orintraperitoneal dosing compared to that of intravenous dosing. Theestimated bioavailability following subcutaneous and intraperitonealinjection were 35.8% and 63.9% respectively. Because only one proteindose was studied, the bioavailability was not evaluated as a function ofdose. The estimated clearance and volume of distribution (based on thedata from the intravenous injection) were 0.48 ml/min. and 6.1 ml,respectively.

Although the data are preliminary, the fate of zalpha11 Ligandadministered IV was similar to that reported for IL-2, another 4-helixbundle cytokine (Donahue, J H and Rosenburg, S A supra.). Like 11-2,IV-administered zalpha11 Ligand had a plasma half life of only minuteswith the main clearance in the kidney. Three hours after injection, themajority of the labeled material extracted from kidney was stillretained in a Biomax 5K NMLW membrane (Millipore). Since it haspreviously been reported that the indium remains associated with proteineven during lysosomal degradation (Staud, F. et al., J. Pharm. Sciences88:577-585, 1999) zalpha11 Ligand is accumulating and may be degraded inthe kidney. The current study also showed, as observed with many otherproteins, including IL-2 (Donahue, J H and Rosenburg, S A, supra.), thatIP and SC administration significantly prolonged the plasma levels ofzalpha11 Ligand.

Example 39 Isolation and Expansion of Fresh Human Bone Marrow MNC CD34+Fraction Using Zalpha11 Ligand for Assessment of NK Activity

A. Selection and Isolation of CD34+ Cells from Human Bone Marrow

Fresh human bone marrow mononuclear cells (MNC) were prepared to enrichfor cells having NK cell activity. Fresh human MNCs were obtained fromPoeitic Technologies (Gaithersburg, Md.). 10 ml alpha MEM (JRH, Lenexa,Kans.) containing 10% HIA FBS (Hyclone, Logan, Utah) and the antibiotic1% PSN (Gibco, BRL, Grand Island, N.Y.) was added to the cell suspensionand the cells were passed through a 100 μm sieve. The cells were thencounted, pelleted, washed with 10 ml PBS containing 2% FBS, thenpelleted again and resuspended in 1 ml PBS containing 2% FBS. Cellshaving a CD34 cell surface marker (CD34+ cells) were magneticallyseparated using a Detachabead kit with Dynabeads M-450 CD34 ((Dynal,Oslo, Norway), as per manufacturer's instructions. Both the CD34+ celland the CD34-cell fractions were further analyzed below.

B. Expansion of CD34+ Cells Using Zalpha11 Ligand

A CD34+ cell fraction was plated into four wells in a 24-well plate.50,000 positively selected cells suspended in 1 ml Alpha MEM (JRH)containing 10% HIA FBS (Hyclone) and 1% PSN (Gibco/BRL), plus thevarious cytokines described below were plated in each of the 4 wells(1-4). Various reagents were used to test for zalpha11 Ligand-inducedexpansion of the CD34+ selected bone marrow MNCs: Reagents includedhuman flt3 (R&D, Minneapolis, Minn.); purified human zalpha11 Ligand(Example 30C and Example 30D); human IL-15 (R&D). Reagents were combinedas follows at day 0: In well #1, 2 ng/ml human flt3 was added. In well#2, 2ng/ml human flt3 and 15 ng/ml purified human zalpha11 Ligand wereadded. In well #3, 2 ng/ml human flt3 and 20 ng/ml human IL15 wereadded. In well #4, 2 ng/ml human flt3, 15 ng/ml purified human zalpha11Ligand, and 20 ng/ml human IL15 were added. After incubating for 18days, the suspension cells from each well were pelleted, and thenresuspended in 0.5 ml alpha MEM (JRH) containing 10% HIA FBS (Hyclone)and 1% PSN (Gibco/BRL), and counted to assess proliferation of the CD34+cell fraction. A low level of proliferation was seen in the presence offlt3 alone (control well #1), but the presence of IL-15 or zalpha11 inaddition to flt3 had not significant effect on the expansion (wells, #2and #3). However, expansion beyond the flt3 control was evident in well#4 which contained IL-15 and zalpha11 Ligand in addition to flt3. Thisresult suggested that zalpha11 and IL-15 act in synergy to expand thehuman CD34+ cell population. Moreover, the results of this experimentsupported the results seen with the mouse zalpha11 Ligand in the mouseBM assay (Example 21).

All cell populations were then tested for NK activity and subjected toflow cytometry analysis, as shown below (Example 41).

C. Expansion of CD34+ or CD34-Cells Using Zalpha11 Ligand With DelayedAddition of IL-15

Both CD34 positive and negative (CD34−) fractions were plated separatelyinto six 12 well plate wells (1-6). Each of six wells contained 100,000positively or negatively selected cells in 2 ml alpha MEM containing 10%FHA FBS and PSN, described above. Reagents used were as described above.In well #1, 2 ng/ml human flt3 was added at day 0. In well #2, 2 ng/mlhuman flt3 was added at day 0, and after 5 days incubation 20 ng/mlhuman IL15 was added. In well #3, 2 ng/ml human flt3 and 15 ng/ml humanzalpha11 Ligand were added at day 0. In well #4, 2 ng/ml human flt3 and15 ng/ml human zalpha11 Ligand were added at day 0, and after 5 daysincubation 20 ng/ml human IL15 was added. In well #5, 2 ng/ml human flt3and 20 ng/ml human IL15 were added at day 0. In well #6, 2 ng/ml humanflt3, 15 ng/ml human zalpha11 Ligand, and 20 ng/ml human IL15 were addedat day 0. After incubating for a total of 15 days from the start of theexperiment, the cells from each well were harvested and counted.

In the CD34+ population a low level of proliferation was seen in thepresence of flt3 alone (control well #1), but the presence of IL-15 orzalpha11 added at day 0 in addition to flt3 had no significant effect onthe expansion (wells, #3 and #5). Addition of IL-15 after 5 days hadsome proliferative effect in comparison to the flt3 control (well #2compared to well #1) and a proliferative effect in the presence ofzalpha11 (well #4 compared to well #3). However, the greatest expansionwas evident in well #6 which contained IL-15 and zalpha11 Ligand inaddition to flt3 at day 0.

In the CD34− population, no proliferation was seen in the presence offlt3 alone (control well #1), and in fact a decrease in the cellpopulation was evident. The presence of zalpha11 added at day 0 inaddition to flt3 (well #3) was similar to the flt3 control. The presenceof IL-15 added at day 5 increased proliferation effect of the cells inthe presence (well #4) or absence (well #2) of zalpha11 Ligand. Again,the greatest expansion was evident in well #6 which contained IL-15 andzalpha11 Ligand in addition to flt3 at day 0.

All cell populations were then tested for NK activity and subjected toFACS analysis, as shown below (Example 41).

Example 40 Isolation and Expansion of Fresh Mouse Cells Using Human andMouse Zalpha11 Ligand for Assessment of NK Activity and NK Cell Markers

A. Isolation and Expansion of Fresh Mouse Low Density Bone Marrow CellsUsing Human and Mouse Zalpha11 Ligand

Fresh mouse marrow cells were isolated by clipping both ends of mousefemurs, and flushing two to three milliliters of growth medium (seebelow) through the inside of the bone into a collection tube. The growthmedium was 500 ml RPMI 1640 Medium (JRH Biosciences. Lenexa, Kans.); 5ml 100× L-glutamine (Gibco BRL. Grand Island, N.Y.); 5 ml 100× NaPyruvate (Gibco BRL); 5 ml 100× Penicillin, Streptomycin, Neomycin (PSN)(Gibco BRL); and 50 ml heat-inactivated Fetal Bovine Serum (FBS)(Hyclone Laboratories. Logan, Utah). The marrow cells were thenbroken-up by pipeting the media up and down several times. The cellswere then pelleted and washed once with growth medium, and passedthrough a 70-micron sieve. The low-density mononuclear cells were thenisolated by subjecting the marrow cells to a density gradient. Marrowcells in five to eight milliliters of growth medium were carefullypipetted on top of five to eight milliliters of NycoPrep 1.077 Animal(Nycomed. Oslo, Norway) in a centrifuge tube. This gradient was thencentrifuged at 600×g for 20 minutes. The low density mononuclear cellswere harvested from the interface layer between the NycoPrep and themedium. These cells were then diluted to approximately 20 milliliters ingrowth medium, pelleted and washed. The cells were then plated atapproximately 0.5-1.5×10⁶ cells per milliliter in growth medium in astandard tissue culture flask and incubated at 37° C., 5% CO₂ for twohours.

The non-adherent, low density (NA LD) marrow cells were then harvestedand plated at 0.5-2.0×10⁵ cells per milliliter in growth medium plus 2.5nanograms per milliliter mouse flt3 (R and D Systems. Minneapolis,Minn.) plus 25 to 50 nanograms per milliliter human Interleukin 15(IL-15) (R and D Systems) with or without 50 to 150 nanograms permilliliter human zalpha11 Ligand; or with or without 0.12 to 10nanograms per milliliter mouse zalpha11 Ligand.

There was no significant expansion without the addition of the human ormouse zalpha11 Ligand. Non-adherent cells were expanded in the culturescontaining mouse zalpha11 Ligand as low as 0.12 ng/ml and in thecultures containing human zalpha11 Ligand as low as 22 ng/ml. Incultures containing both the human and mouse zalpha11 Ligand,non-adherent cell expansion increased with increasing dose if zalpha11Ligand, with the mouse ligand saturating response at about 5-10 ng/mland the human not reaching a saturating response even at the highestdose of 200 ng/ml. Human zalpha11 Ligand appeared to be approximately 20to 100 fold less potent on mouse cells as the mouse zalpha11 Ligand.After approximately five to ten days the zalpha11 Ligand expanded mousecells were harvested and analyzed by flow cytometry (FACSCalibur; BectonDickinson, Mansfield, Mass.) to determine what percentage of them werepositive for NK cell antigens, where 46% were positive for the PanNKcell marker DX5 (Pharmingen).

B. Isolation and Expansion of Fresh Lineage Depleted Mouse Marrow Cells

Fresh mouse lineage depleted (lin−) marrow cells were isolated fromfresh mouse marrow cells by first incubating the cells with thefollowing antibodies: TER119, Gr-1, B220, MAC-1, CD3e and 1-Ab(Pharmingen. San Diego, Calif.). The lin+ cells were then removed withDynabeads M-450 sheep anti-rat IgG (Dynal, Lake Success, N.Y.) as permanufacturer's instructions.

The negatively selected lin− marrow cells were then plated as above ingrowth medium plus either 2.5 ng/mL flt3(R&D Systems) and 25 ng/mL IL-15(R&D Systems); or flt3, IL-15 and mouse zalpha11 Ligand, 2 to 5% BHKmouse zalpha11 Ligand conditioned medium. After six days of growth, thecultures were harvested, counted and submitted to an NK cell activityassay (Example 41). Cells grown with mouse zalpha11 Ligand wereapproximately two to three times more effective at lysing NK cell targetcells (YAC-1 cells) as the cells grown without zalpha11 Ligand.

C. Isolation and Expansion of CD4− CD8− (Double Negative or DN)Thymocvtes

Fresh mouse thymocytes were isolated by chopping and sieving thymusesfrom three to eight week old mice. CD4− CD8− (DN) cells were thennegatively selected by incubating the thymocytes with anti-CD4 andanti-CD8 antibodies (PharMingen), then removing the CD4+ CD8+ cells withDynabeads M-450 sheep anti-rat IgG (Dynal) as per manufacturer'sinstructions.

The DN mouse thymocytes were then grown in growth medium plus 2.5 ng/mLflt3 (R&D Systems), 25 ng/mL IL-15 (R&D Systems) and 10 ng/mL IL-7 (R&DSystems) with or without mouse zalpha11 Ligand as above. Six days laterthe cells were harvested, counted, analyzed by flow cytometry asdescribed above, and also submitted to an NK cell activity assay(Example 41).

The culture grown with mouse zalpha11 Ligand yielded approximately480,000 cells while the culture without zalpha11 Ligand yielded onlyapproximately 160,000 cells. The culture grown with mouse zalpha11Ligand was found to be approximately 16.2% positive for the NK cellantigen Pan NK, DX5 (PharMingen). The culture grown without zalpha11Ligand was 14.6% positive for DX5. The cells grown with zalpha11 Ligandlysed NK cell target cells, YAC-1, approximately two times better thanthe cells grown without zalpha11 Ligand. The expanded cells did not lysesignificantly a negative control target cell line, EL4. These resultssuggested that zalpha11 Ligand selectively expands lytic NK cells.

Example 41 Activity of Human and Mouse Zalpha11 Ligand Expanded Cellsand Mature Murine NK Cells in NK Cell Cytotoxicity Assays

A. NK Cell Assay

NK cell-mediated target cytolysis was examined by a standard⁵¹Cr-release assay. Target cells (K562 cells (ATCC No. CCL-243) in humanassays, and YAC-1 cells (ATCC No. TIB-160) in mouse assays) lackexpression of major histocompatability complex (MHC) molecules,rendering them susceptible to NK cell-mediated lysis. A negative controltarget cell line in mouse assays is the MHC⁺ thymoma EL4 (ATCC No.TIB-39). We grew K562, EL4, and YAC-1 cells in RP10 medium (standardRPMI 1640 (Gibco/BRL, Grand Island, N.Y.) supplemented with 10% FBS(Hyclone, Logan, Utah), as well as 4 mM glutamine (Gibco/BRL), 100I.U./ml penicillin+100 MCG/ml streptomycin (Gibco/BRL), 50 μMβ-mercaptoethanol (Gibco/BRL) and 10 mM HEPES buffer (Gibco/BRL). On theday of assay, 1-2×10⁶ target cells were harvested and resuspended at2.5-5×10⁶ cells/ml in RP10 medium. We added 50-100 μl of 5mCi/ml⁵¹Cr-sodium chromate (NEN, Boston, Mass.) directly to the cellsand incubated them for 1 hour at 37° C., then washed them twice with 12ml of PBS and resuspended them in 2 ml of RP10 medium. After countingthe cells on a hemacytometer, the target cells were diluted to 0.5-1×10⁵cells/ml and 100 μl (0.5-1×10⁴ cells) were mixed with effector cells asdescribed below.

In human assays, effector cells were prepared from selected and expandedhuman CD34+ BM cells (Example 39B) which were harvested, washed,counted, mixed at various concentrations with ⁵¹Cr-labeled target cellsin 96-well round bottomed plates, and incubated for 4 hours at 37° C.After co-incubation of effector cells and the labeled target cells, halfof the supernatant from each well was collected and counted in a gammacounter for 1 min/sample. The percentage of specific ⁵¹Cr release wascalculated from the formula 100×(X-Y)/(Z-Y), where X is ⁵¹Cr release inthe presence of effector cells, Y is the spontaneous release in theabsence of effectors, and Z is the total ⁵¹Cr release from target cellsincubated with 0.5% Triton X-100. Data were plotted as the % specificlysis versus the effector-to-target ratio in each well.

B. Activity of Human Zalpha11 Ligand Expanded Cells

Isolated CD34+ human HPCs cultured with flt3±zalpha11 Ligand andflt3+IL-15±zalpha11 Ligand (Example 39), were harvested the cells on day15 to assess their capacity to lyse MHC⁻ K562 cells in a standard⁵¹Cr-release assay as described above, and to analyze their surfacephenotype by flow cytometry. As expected from previous reports (Mrozek,E et al., Blood 87:2632-2640, 1996; and Yu, H et al., Blood92:3647-3657, 1998), simultaneous addition of IL-15 and flt3L did inducethe outgrowth of a small population of CD56⁺ cells. Interestingly,although BM cells cultured simultaneously with zalpha11 Ligand and flt3Ldid not expand significantly, there was a significant increase in totalcell numbers in cultures containing a combination of flt3L, zalpha11Ligand and IL-15 (see, Example 39).

For an assessment of the surface phenotype of these human BM cultures,we stained small aliquots of the cells for 3-color flow cytometricanalysis with anti-CD3-FITC, anti-CD56-PE and anti-CD16-CyChrome mAbs(all from PharMingen, San Diego, Calif.) and analyzed them on aFACSCalibur using CellQuest software (Becton Dickinson, Mountain View,Calif.). This flow cytometric analysis confirmed that the cells growingout of these cultures were differentiated NK cells, as they were largeand granular and expressed both CD56 and CD16, and were CD3⁻ (Lanier, LL Annu. Rev. Immunol. 16:359-393, 1998). Furthermore, these cellsexhibited significantly higher effector function than those cells grownwith IL-15 and flt3. More specifically, cells grown in all threecytokines lysed more than 40% of the K562 targets at aneffector-to-target ratio (E:T) of 1.5, whereas cells grown inIL-15+flt3L lysed fewer than 5% of the targets at an E:T of 2. Thesedata demonstrate that, in combination with IL-15, zalpha11 Ligandstimulates the differentiation of NK cells from CD34⁺ BM cells.

C. Activity of Mouse Zalpha11 Ligand Expanded Cells

To test the effects of zalpha11 Ligand on murine hematopoieticprogenitor cells, purified Lineage-negative (Lin−) bone marrow cellsfrom C57B1/6 mice were expanded in flt3+IL-15±zalpha11 Ligand, asdescribed in Example 40B. On day 6 of culture, the cells (“effectors”)were harvested and counted, then resuspended in 0.4 ml of RP10 medium(Example 41A). Two aliquots (0.15 ml each) of each sample expanded withor without zalpha11 Ligand (Example 41A) were diluted serially 3-fold induplicate in 96-well round bottomed plates, for a total of 6 wells of100 μl each. The remaining 100 μl of cells were stained for NK cellsurface markers with FITC-anti-2B4 and PE-anti-DX5 mAbs (PharMingen) andanalyzed by flow cytometry. Each group of cells exposed to flt3+IL-15with or without the presence of zalpha11Ligand had similar fractions of2B4+DX5+ cells, ranging from 65-75% positive for both NK markers.

For the NK lysis assay, target cells (YAC-1 and EL4) were labeled with⁵¹Cr as described above. After counting the target cells on ahemacytometer, the target cells were diluted to 0.5-1×10⁵ cells/ml and100 μl of YAC-1 or EL4 (0.5×10⁴ cells) were mixed with 100 μl effectorcells and incubated for 4 hours at 37° C. Specific lysis was determinedfor each well as described above.

We found that cells grown in the presence of flt3+IL-15+zalpha11 Ligandexhibited enhanced lytic activity (roughly 2-fold) against the YAC-1targets (but did not kill the MHC⁺ control cell line EL4). At aneffector-to-target ratio (E:T) of 5, NK cells generated in the presenceof all 3 cytokines (zalpha11 Ligand+flt3+IL-15) lysed 12% of the YAC-1cells, whereas those NK cells expanded with flt3+IL-15 lysed 6% of theYAC-1 targets. Subsequent experiments confirmed this trend.

In a second approach to determine the biological activity of zalpha11Ligand on murine NK cells, we isolated immature CD4⁻CD8⁻(“doublenegative”, DN) mouse thymocytes as described in Example 40C and culturedthem with IL-15+flt3+IL-7 or IL-15+flt3+IL-2, with or without zalpha11Ligand. On day 6 of culture, the cells were harvested and assayed for NKlytic activity on YAC-1 and EL4 cells as described above. We found thatcells cultured in the presence of zalpha11 Ligand had the greatest lyticactivity in this assay, with enhanced lytic activity over those cellscultured in the presence of the other cytokines. Specifically, DNthymocytes grown with IL-15+flt3+IL-7 killed 18% of the YAC-1 cells atE:T of 24 while cells grown in the presence of IL-15+flt3+IL-7 pluszalpha11 Ligand killed 48% of the targets at the same E:T. DN thymocytesgrown in IL-15+flt3+IL-2 killed 15% of the YAC-1 targets at an E:T of 6,whereas cells grown with these 3 cytokines and zalpha11 Ligand killed35% of the YAC-1 cells at an E:T of 9. Flow cytometry was performed onthe cultured cells one day before the NK lysis assay. As was true forthe bone marrow cultures, despite the proliferative effect of zalpha11Ligand (cell numbers increase approximately 2-fold when zalpha11 Ligandis added), it did not significantly enhance the fraction of DX5⁺ cells(17-20% of total cells in the cultures with IL-7, and 35-46% of total incultures with IL-2). These data imply that zalpha11 Ligand, incombination with IL-15 and flt3, enhances the lytic activity of NK cellsgenerated from murine bone marrow or thymus.

D. Activity of Mouse Zalpha11 Ligand on Mature Murine NK Cells

In order to test the effects of mouse zalpha11 Ligand on mature NKcells, we isolated spleens from four 5-week old C57B1/6 mice (JacksonLaboratories, Bar Harbor, Me.) and mashed them with frosted-end glassslides to create a cell suspension. Red blood cells were removed byhypotonic lysis as follows: cells were pelleted and the supernatantremoved by aspiration. We disrupted the pellet with gentle vortexing,then added 900 μl of sterile water while shaking, followed quickly (lessthan 5 sec later) by 100 μl of 10×HBSS (Gibco/BRL). The cells were thenresuspended in 10 ml of 1×HBSS and debris was removed by passing thecells over a nylon mesh-lined cell strainer (Falcon). These RBC-depletedspleen cells were then pelleted and resuspended in MACS buffer (PBS+1%BSA+2 mM EDTA) and counted. We stained 300×10⁶ of the cells withanti-DX5-coated magnetic beads (Miltenyi Biotec) and positively selectedDX5⁺ NK cells over a MACS VS+ separation column, according to themanufacturer's instructions, leading to the recovery of 8.4×10⁶ DX5⁺cells and 251×10⁶ DX5⁻ cells. Each of these groups of cells werecultured in 24-well plates (0.67×10⁶ cells/well, 2 wells per treatmentcondition) in RP10 medium (Example 41A) alone or with 1) 30 ng/ml mousezalpha11 Ligand, 2) 30 ng/ml recombinant mouse IL-2 (R&D Systems, Inc.,Minneapolis, Minn.), 3) 30 ng/ml recombinant human IL-15 (R&D), 4) 30ng/ml each of mouse zalpha11 Ligand and hIL-15, or 5) 30 ng/ml each ofmIL-2 and hIL-15. The cells were harvested after 21 hours, washed, andresuspended in RP10 medium and counted. The cells were then assayed fortheir ability to lyse ⁵¹Cr-labeled YAC-1 or EL4 targets cells, asdescribed in Example 41A.

In general, there was little NK activity from the DX5⁻ (non-NK cells)groups, but the DX5⁻ cells cultured with zalpha11 Ligand and hIL-15 didlyse 25% of the YAC-1 target cells at an E:T of 82. By comparison, DX5⁻cells cultured with hIL-15 alone lysed 14% of the YAC-1 targets at anE:T of 110. This suggests that zalpha11 Ligand and IL-15 are actingtogether on the residual NK1.1⁺ NK cells in this cell preparation. Asfor the DX5⁺ cell preparation, treatment with mouse zalpha11 Ligandalone did not significantly increase their effector function (theirlysis of YAC-1 cells was similar to the untreated group). As expected,both IL-2 and IL-15 significantly improved NK activity. The highestlevel of lysis, however, was detected in the group treated with zalpha11Ligand and hIL-15 (65% lysis of YAC-1 cells at an E:T of 3.3, vs. 45%lysis at an E:T of 4 for the hIL-15 treatment group). Taken together,these results suggest that although zalpha11 Ligand alone may notincrease NK cell lysis activity, it does enhance NK lysis activity ofmature NK cells, when administered with IL-15.

Example 42 Zalpha11 Ligand Proliferation of Human and Mouse T-cells in aT-cell Proliferation Assay

A. Murine Zalpha11 Ligand Proliferation of Mouse T-cells

T cells from C57B1/6 mice (Jackson Laboratories, Bar Harbor, Me.) wereisolated from pooled splenocytes and lymphocytes from axillary,brachial, inguinal, cervical, and mesenteric lymph nodes (LNs). Spleenswere mashed with frosted-end glass slides to create a cell suspension.LNs were teased apart with forceps and passed through a cell strainer toremove debris. Pooled splenocytes and LN cells were separated into CD8⁺and CD4⁺ subsets using two successive MACS magnetic separation columns,according to the manufacturer's instructions (Miltenyi Biotec, Auburn,Calif.). Whole thymocytes were collected from the same mice.

Cells were cultured at 3×10⁵ cells/well (thymocytes) or 10⁵ cells/well(mature T cells) with increasing concentrations of purified murinezalpha11 Ligand (0-30 ng/ml) (Example 24 and Example 29) in 96-well flatbottomed plates pre-coated overnight at 4° C. with variousconcentrations of anti-CD3 mAb 2C11 (PharMingen) for 3 days at 37° C.The anti-CD3 antibody served to activate the murine T-cells through theT-cell receptor. Each well was pulsed with 1μCi ³H-thymidine on day 2and plates were harvested and counted 16 hours later to assessproliferation.

When we tested zalpha11 Ligand in T cell proliferation assays, we foundthat it co-stimulated anti-CD3-activated murine thymocytes, leading toan accelerated outgrowth of CD8⁺CD4⁻ cells (the majority of thethymocytes cultured with anti-CD3+zalpha11 Ligand were CD8⁺CD4⁻ by day 3of culture, while cells cultured with anti-CD3 alone did notsignificantly skew to this phenotype until day 5). We did not observesignificant levels of proliferation of thymocytes to zalpha11 Ligand inthe absence of anti-CD3.

Interestingly, when we assayed mature peripheral murine T cells fortheir ability to respond to zalpha11 Ligand+anti-CD3, we found that onlythe CD8⁺, but not the CD4⁺ subset, responded in a dose-dependent mannerto zalpha11. Ligand. We also observed weak but reproducibleproliferation of CD8⁺ cells (but not CD4⁺ cells) in response to zalpha11Ligand alone. Interestingly, this was not observed for human T cells(see Example 42B, below).

B. Human Zalpha11 Ligand Proliferation of Human T-cells

Human CD4+ and CD8+ T cells were isolated from PBMC as described inExample 43 (below) Cells were cultured at about 10⁵ cells/well withincreasing concentrations of purified human zalpha11 Ligand (0-50 ng/ml)(Example 24) in 96-well flat bottomed plates pre-coated overnight at 4°C. with various concentrations of anti-human CD3 mAb UCHT1 (PharMingen)for 3 days at 37° C. Each well was pulsed with 1uCi ³H-thymidine on day2 and plates were harvested and counted 16 hours later. Unlike ourresults with mouse T cells, our preliminary data suggests that humanzalpha11 Ligand co-stimulates CD4+, but not CD8+, human T cells in adose-dependent fashion.

Example 43 Real Time PCR Shows Zalpha11 Ligand Expression in Human CD4+Cells

A. Purified Human T Cells as a Primary Source Used to Assess HumanZalpha11 Ligand Expression

Whole blood (150 ml) was collected from a healthy human donor and mixed1:1 with PBS in 50 ml conical tubes. Thirty ml of diluted blood was thenunderlayed with 15 ml of Ficoll Paque Plus (Amersham Pharmacia Biotech,Uppsala, Sweden). These gradients were centrifuged 30 min at 500 g andallowed to stop without braking. The RBC-depleted cells at the interface(PBMC) were collected and washed 3 times with PBS. The isolated humanPBMC yield was 200×10⁶ prior to selection described below.

The PBMCs were suspended in 1.5 ml MACS buffer (PBS, 0.5% EDTA, 2 mMEDTA) and 3×10⁶ cells were set aside for control RNA and for flowcytometric analysis. We next added 0.25 ml anti-human CD8 microbeads(Miltenyi Biotec) and the mixture was incubated for 15 min at 4° C.These cells labeled with CD8 beads were washed with 30 ml MACS buffer,and then resuspended in 2 ml MACS buffer.

A VS+ column (Miltenyi) was prepared according to the manufacturer'sinstructions. The VS+ column was then placed in a VarioMACS magneticfield (Miltenyi). The column was equilibrated with 5 ml MACS buffer. Theisolated primary mouse cells were then applied to the column. The CD8negative cells were allowed to pass through. The column was rinsed with9 ml (3×3 ml) MACS buffer. The column was then removed from the magnetand placed over a 15 ml falcon tube. CD8+ cells were eluted by adding 5ml MACS buffer to the column and bound cells flushed out using theplunger provided by the manufacturer. The yield of CD8+ selected humanperipheral T cells was about 51×10 ⁶ total cells. The CD8-negative flowthrough cells were collected, counted, stained with anti-human CD4coated beads, then incubated and passed over a new VS+ column at thesame concentrations as described above. The yield of CD4+ selected humanperipheral T cells was 42×10⁶ total cells.

A sample of each of the CD8+ and CD4+ selected human T cells was removedfor staining and sorting on a fluorescence activated cell sorter (FACS)to assess their purity. A PE-conjugated anti-human CD4 antibody, ananti-human CD8-FITC Ab, and an anti-human CD19-CyChrome Ab (all fromPharMingen) were used for staining the CD8+ and CD4+ selected cells. TheCD8-selected cells in this first experiment were 80% CD8+, and theCD4-selected cells were 85% CD4+. In 2 subsequent experiments (Example43B), the CD8+ purified cells were 84% and 81% pure, and the CD4+ cellswere 85% and 97% pure, respectively. In one experiment, we stained thenon-binding (flow-through) cells with anti-human CD19-coated beads(Miltenyi) and ran them over a third magnetic bead column to isolateCD19+ B cells (these were 92% pure).

The human CD8+, CD4+ and CD19+ selected cells were activated byincubating 0.5×10⁶ cells/ml in RPMI+5% human ultraserum (GeminiBioproducts, Calabasas, Calif.)+PMA 10 ng/ml and Ionomycin 0.5 μg/ml(Calbiochem) for about 4, 16, or 24 hours at 37° C. The T-cells(2.5×10⁶/well) were alternately stimulated in 24-well plates pre-coatedovernight with 0.5 μg/ml plate-bound anti-CD3 mAb UCHT1 (PharMingen)with or without soluble anti-CD28 mAb (PharMingen) at 5 μg/ml. At eachtimepoint, the cells were harvested, pelleted, washed once with PBS, andpelleted again. The supernatant was removed and the pellets weresnap-frozen in a dry ice/ethanol bath, then stored at −80° C. for RNApreparation at a later date.

Real Time-PCR was performed on these human CD8+, CD4+ and CD19+ selectedcells as described in Example 43B and 43C below for assessing humanzalpha11 Ligand and receptor expression.

B. Primers and Probes for Quantitative RT-PCR for Human Zalpha11 LigandExpression

Real-time quantitative RT-PCR using the ABI PRISM 7700 SequenceDetection System (PE Applied Biosystems, Inc., Foster City, Calif.) hasbeen previously described (see, Heid, C A et al., Genome Research6:986-994, 1996; Gibson, U E M et al., Genome Research 6: 995-1001,1996; and Sundaresan, S et al., Endocrinology 139:4756-4764, 1998). Thismethod incorporates use of a gene specific probe containing bothreporter and quencher dyes. When the probe is intact the reporter dyeemission is negated due to the proximity of the quencher dye. During PCRextension using additional gene-specific forward and reverse primers,the probe is cleaved by 5′ nuclease activity of Taq polymerase whichreleases the reporter dye resulting in an increase in fluorescentemission.

The primers and probes used for real-time quantitative RT-PCR analyseswere designed using the primer design software Primer Express™ (PEApplied Biosystems). Primers for human zalpha11 Ligand were designedspanning an intron-exon junction to eliminate amplification of genomicDNA. The forward primer, ZC22,281 (SEQ ID NO:90) and the reverse primer,ZC22,279 (SEQ ID NO:91) were both used at 300 nM concentration tosynthesize an 80 bp product. The corresponding zalpha11 Ligand TaqManprobe, ZG32 (SEQ ID NO:92) was synthesized by PE Applied Biosystems. Theprobe was labeled with a reporter fluorescent dye(6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) at the 5′ end anda quencher fluorescent dye (6-carboxy-tetramethyl-rhodamine) (TAMRA) (PEApplied Biosystems) at the 3′ end. In order to test the integrity orquality of all the RNA samples, they were screened for rRNA using theprimer and probe set ordered from PE Applied Biosystems (cat No.4304483). The reporter fluorescent dye for this probe is VIC (PE AppliedBiosystems). The rRNA results will allow for the normalization of thezalpha11 Ligand results.

RNA was prepared from pellets provided in Example 43A, using RNeasyMiniprep™ Kit (Qiagen, Valencia, Calif.) per the manufacturer'sinstructions. Control RNA was prepared from about 10 million BHK cellsexpressing human zalpha11 Ligand.

C. Primers and Probes for Quantitative RT-PCR for Human Zalpha11Receptor Expression

Real time PCR was performed to assess the expression of zalpha11receptor as per Example 43B and Example 43D, using the cells preparedunder the conditions detailed in 43A, and probes specific for thezalpha11 receptor. The forward primer, ZC22,277 (SEQ ID NO:93) and thereverse primer, ZC22,276 (SEQ ID NO:94) were used in a PCR reaction(above) at about 300 nM concentration to synthesize a 143 bp product.The corresponding zalpha11 TaqMan® probe, designated ZG31 (SEQ ID NO:95)was synthesized and labeled by PE Applied Biosystems. RNA from BaF3cells expressing human zalpha11 receptor was used to generateappropriate control for standard curves for the real-time PCR describedin Example 43D below.

D. Real-Time Quantitative RT-PCR

Relative levels of zalpha11 Ligand RNA were determined by analysis oftotal RNA samples using the One-Step RT-PCR method (PE AppliedBiosystems). RNA from BHK cells expressing human zalpha11 Ligand wasused to generate a standard curve. The curve consisted of serialdilutions ranging from 2.5-2.5×10⁴ ng for the rRNA screen and 25-0.0025ng for the zalpha11 Ligand screen with each point analyzed intriplicate. The total RNA samples were also analyzed in triplicate forhuman zalpha11 Ligand transcript levels and for levels of rRNA as anendogenous control. Each One-step RT-PCR reaction consisted of 25 ng oftotal RNA in buffer A (50 mM KCL, 10 mM Tris-HCL, and the internalstandard dye, ROX (PE Applied Biosystems)), appropriate primers (50 nMfor rRNA samples, 300 nM for zalpha11 Ligand samples) and probe (50 nMfor rRNA, 100 nM for zalpha11 Ligand), 5.5 mM MgCl₂, 300 μM each d-CTP,d-ATP, and d-GTP and 600 μM of d-UTP, reverse transcriptase (0.25 U/μl),AmpliTaq DNA polymerase (0.025 U/μl) and RNase Inhibitor (0.4 U/μl) in atotal volume of 25 μl. Thermal cycling conditions consisted of aninitial RT step at 48° C. for 30 minutes, an AmpliTaq Gold activationstep of 95° C. for 10 minutes, followed by 40 cycles of amplificationfor 15 seconds at 95° C. and 1 minute at 60° C. Relative zalpha11 LigandRNA levels were determined by the Standard Curve Method as described inUser Bulletin No. 2 (PE Biosystems; User Bulletin #2: ABI Prism 7700Sequence Detection System, Relative Quantitation of Gene Expression,Dec. 11, 1997) using the rRNA measurements to normalize the zalpha11Ligand levels. Samples were compared relative to the calibrator withineach experiment. The calibrator was arbitrarily chosen based on goodquality RNA and an expression level to which other samples couldsignificantly be compared. Results of the experiments analyzing theexpression of the zalpha11 Ligand and zalpha receptor in stimulated andunstimulated cells (Example 43A) are as described in Example 43E below.

E. Expression of Human Zalpha11 Receptor and Ligand in CD4+, CD8+ andCD19+Cells

The first experiment used RT-PCR, described above, to assess zalpha11receptor expression in unstimulated and anti-CD3 stimulated CD4+ andCD8+ samples at timepoints of 0 h (unstimulated (“resting”) cells), andat 4 h, 15.5 h and 24 h, after stimulatoin. The resting CD4+ sample wasarbitrarily chosen as the calibrator and given a value of 1.00. Therewas approximately a 4-fold increase in receptor expression inunstimulated CD4+ cells from 4 h to 24 h of culture and about an 8-foldincrease over the same time period in anti-CD3 stimulated CD4+ cells.The CD8+ cells showed a 7-fold increase in zalpha11 receptor expressionthat peaked at 4 hrs and decreased over time. With anti-CD3 stimulation,the CD8+ cells had a constant 8-fold increase in receptor expression.

This first experiment also used RT-PCR to assess zalpha11 Ligandexpression in the same anti-CD3 stimulated and unstimulated CD4+ andCD8+ samples. The 4 hr anti-CD3 stimulated CD8+ sample was arbitrarilychosen as the calibrator and given a value of 1.00. The results showedthat unstimulated CD4+ and CD8+ cells do not express zalpha11 Ligand. Weobserved a significant elevation of expression in the anti-CD3stimulated CD4+ cells at 4 h, with about a 300-fold increase in signalobserved at 15.5 h. The CD8+ cells expressed a small amount of ligandupon anti-CD3 stimulation, however this is probably due to contaminationof the CD8+ population with a small number of CD4+ cells.

The second experiment used RT-PCR to assess zalpha11 receptor expressionin anti-CD3-stimulated, PMA+Ionomycin-stimulated and unstimulated CD4+and CD8+ samples at timepoints of 0 h, and at 3.5 h, 16 h and 24 h afteractivation. The resting CD8+ sample was arbitrarily chosen as thecalibrator and given a value of 1.00. The resting CD4+ and CD8+ cellsdid not have significant amounts of receptor expression. The expressionwas about 3 fold higher in the PMA+Ionomycin-stimulated CD4+ samples at3.5 h, 16 h and 24 h after stimulation. The expression in anti-CD3activated CD4+ cells peaked at 10-fold above background levels at 3.5 hafter stimulation, then fell back to levels 4-fold above background at16 h after stimulation. The CD8+ cells showed a 4-fold expressionincrease at 3.5 h after PMA+Ionomycin stimulation, with expressiondecreasing at subsequent timepoints. As in the first experiment, theanti-CD3 stimulated CD8+ cells again exhibited an 8-fold abovebackground induction of receptor expression.

These samples from the second experiment were also used to assesszalpha11 Ligand expression. The 24hr PMA + Ionomycin stimulated CD4+sample was arbitrarily chosen as the calibrator and given a value of1.00. The results showed that again none of the unstimulated cellsexpressed zalpha11 Ligand. There was about a 30-fold induction of ligandexpression in the CD4+ cells stimulated with anti-CD3 at 3.5 h, as seenin the previous experiment (at 4 h). However, there was only about a5-fold induction with PMA+Ionomycin stimulation at 3.5 h that went downat subsequent timepoints. Again, the CD8+ cells expressed a very smallamount of Ligand that was probably attributed to contaminating CD4+cells.

The final experiment used RT-PCR to assess zalpha11 receptor expressionin anti-CD3- and anti-CD3/anti-CD28-stimulated and unstimulated CD4+ andCD8+ samples at timepoints of 0 h, and at 2 h, 4 h, and 16 h afterstimulation. CD19+ cells activated with PMA+Ionomycin were also screenedfor receptor expression at the same time intervals. The resting CD4+sample was arbitrarily chosen as the calibrator and given a value of1.00. The 2 h anti-CD3 stimulated CD4+ cells only had a 4-fold inductionof receptor, compared to the 10-fold induction seen at 3.5 h in theprevious experiment. The combination of anti-CD3 and anti-CD28 increasedzalpha11 receptor expression to 8-fold above background. The 16 hanti-CD3/anti-CD28 stimulated CD8+ cells had very low zalpha11 receptorexpression levels, as seen in the CD8+ cells in previous experiments(above). The CD19+ cells stimulated with PMA+Ionomycin had the mostsignificant zalpha11 receptor expression with a 19-fold increase at 2 h,but the expression levels decreased back to those of resting cells by 16h.

These samples from the final experiment were also used to assesszalpha11 Ligand by RT-PCR. The 16 h anti-CD3/anti-CD28 stimulated CD8+sample was arbitrarily chosen as the calibrator and given a value of1.00. The results showed that at 2 h the CD4+ cells had about a 2-foldinduction of zalpha11 Ligand expression with anti-CD3 stimulation and a5-fold induction with anti-CD3 plus anti-CD28 stimulation. Thesestimulation conditions induced Ligand expression over time, with the 16h stimulated CD4+ cells exhibiting Ligand expression levels 70-foldabove background. CD8+ and CD19+ cells showed no zalpha11 Ligandexpression.

A certain amount of variation was expected between blood draws (i.e.multiple samples at different times from the same patient and betweenmultiple patients). Therefore, data trends were analyzed within eachstudy or from a single blood sample and the three experiments above werecompared for an overall conclusion. The trend from the Real Time PCRexperiments described above is that of all the cell types tested, CD19+B cells activated with PMA+ionomycin expressed the highest levels ofzalpha11 receptor RNA. CD4+ and CD8+ cells can also be stimulated toexpress receptor, but at lower levels than in B cells. Zalpha11 Ligandwas expressed almost exclusively in stimulated CD4+ T cells (and not byCD8+ T cells or CD19+ B cells). Although stimulation with PMA+Ionomycininduced a good zalpha11 Ligand signal in this assay, a significantlyhigher signal was obtained from CD4+ T cells stimulated with anti-CD3mAb or a combination of anti-CD3 and anti-CD28 mAbs, conditions thatbetter mimic an antigen encounter in vivo.

Example 44 Zalpha11 Ligand-Dependent Proliferation of B-Cell CellsStimulated Anti-CD40 or Anti-IgM

A. Purification of Human B Cells

A vial containing 1×10⁸ frozen, apheresed human peripheral bloodmononuclear cells (PBMCs) was quickly thawed in a 37 ° C. water bath andresuspended in 25 ml B cell medium (RPMI Medium 1640 (JRH Biosciences.Lenexa, Kans.), 10% Heat inactivated fetal bovine serum, 5% L-glutamine,5% Pen/Strep) (Gibco BRL)) in a 50 ml tube (Falcon VWR, Seattle, Wash.).Cells were tested for viability using Trypan Blue (Gibco BRL). Tenmilliliters of Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology Inc.,Piscataway, N.J.) was layered under the cell suspension and spun for 30minutes at 1800 rpm and allowed to stop with the brake off. Theinterface was then removed and transferred to a fresh 50 ml Falcon tube,brought up to a final volume of 40 ml with PBS and spun for 10 minutesat 1200 rpm with the brake on. The viability of the isolated cells wasagain tested using Trypan Blue. Alternately fresh drawn human blood wasdiluted 1:1 with PBS (Gibco BRL) and layered over Ficoll/Hypaque Plus(Pharmacia), spun and washed as above. Cells isolated from either freshor frozen sources gave equivalent results.

B cells were purified from the Ficoll floated peripheral blood cells ofnormal human donors (above) with anti-CD19 magnetic beads (MiltenyiBiotec, Auburn, Calif.) following the manufacturer's instructions. Thepurity of the resulting preparations was monitored by flow cytometricanalysis with anti-CD22 FITC Ab (Pharmingen, SanDiego, Calif.). B cellpreparations were typically >90% pure.

B. Purification of Murine B Cells

A suspension of murine splenocytes was prepared by teasing adult C57B1/6mouse (Charles River Laboratories, Wilmington, Mass.) spleens apart withbent needles in B cell medium. RBCs were removed by hypotonic lysis.CD43 positive cells were removed with CD43 magnetic beads (MiltenyiBiotec) following the manufacturer's instructions. The purity of theresulting preparations was monitored by flow cytometric analysis withanti-CD45R FITC Ab (Pharmingen). B cell preparations were typically >90%pure.

C. Proliferation of Anti-CD40-Stimulated B-Cells in the Presence ofHuman or Murine Zalpha11 Ligand

The B cells from either the human or mouse source were resuspended at afinal concentration of ×10⁶ cells/ml in B cell medium and plated at 100μl/well in a 96 well U bottom plate (Falcon, VWR) containing variousstimulation conditions to bring the final volume to 200 μl/well. Foranti-CD40 stimulation human cultures were supplemented with 1 μg/mlanti-human CD40 (Genzyme, Cambridge, Mass.) and mouse cultures weresupplemented with 1 μg/ml anti-murine CD40 (Serotec, UK). Human ormurine zalpha11 Ligand was added at dilutions ranging from 1 pg/ml-100ng/ml. The specificity of the effect of zalpha11 Ligand was confirmed byinhibition of zalpha11 Ligand with 25mg/ml soluble human zalpha11CEE(Example 10A). All treatments were performed in triplicate. The cellswere then incubated at 37° C. in a humidified incubator for 120 hours(human) or 72 hours (mouse). Sixteen hours prior to harvesting, 1 μCi³H-thymidine (Amersham, Piscataway, N.J.) was added to all wells toassess whether the B-cells had proliferated. The cells were harvestedinto a 96 well filter plate (UniFilter GF/C, Packard, Meriden, Conn.)using a cell harvester (Packard) and collected according tomanufacturer's instructions. The plates were dried at 55 ° C. for 20-30minutes and the bottom of the wells were sealed with an opaque platesealer. To each well was added 0.25 ml of scintillation fluid(Microscint-O, Packard) and the plate was read using a TopCountMicroplate Scintillation Counter (Packard).

Incubation with Zalpha11 Ligand at concentrations of 3 ng/ml or moreenhanced the proliferation induced by soluble anti-CD40 in a dosedependent manner in both murine and human B cells by as much as 30 fold.The murine and human B cells responded equally as well to theirrespective zalpha11 Ligand. In both species, the stimulation wasspecific to zalpha11 Ligand, as it was reversed by the presence ofsoluble zalpha11 receptor in the culture.

D. Proliferation of Anti-IgM-Stimulated B-Cells in the Presence of Humanor Murine Zalpha11 Ligand

The B cells from either human or mouse source as described above(Example 44A and Example 44B) were plated as described above (Example44C). For anti-IgM stimulation of human cells the plates were pre-coatedovernight with 10 mg/ml F(ab′)₂ anti-human IgM Abs (Southern BiotechAssociates, Birmingham, Ala.) and washed with sterile media just priorto use. The cultures were supplemented with 0-10 ng/ml hu rIL-4 (R&DSystems, Minneapolis, Minn.). For anti-IgM stimulation of murine cellssoluble anti-IgM (Biosource, Camarillo, Calif.) was added to thecultures at 10 mg/mi. To each of the preceding anti-IgM/IL-4 conditions,human or murine Zalpha11 ligand was added-at dilutions ranging from 1pg/ml-100 ng/ml as described above. The specificity of the effect ofzalpha11 Ligand was confirmed by inhibition with soluble human zalpha11receptor as described above (Example 44C). All treatments were performedin triplicate. The cells were incubated, labeled with ³H-thymidine,harvested, and analyzed as described in Example 44C.

Incubation with Zalpha11 ligand at concentrations of 0.3 ng/ml or moreinhibited the proliferation induced by insoluble anti-IgM (mouse) oranti-IgM and IL-4 (human) in a dose-dependent manner. This inhibitionwas specific to zalpha11 Ligand, as it was reversed by the presence ofsoluble zalpha11 receptor in the culture.

Example 45 Expression of Human Zalpha11 Soluble Receptor in E. coli

A. Construction of Expression Vector pCZR225 That Expresses Huzalpha11/MBP-6H Fusion Polypeptide

An expression plasmid containing a polynucleotide encoding a humanzalpha11 soluble receptor fused C-terminally to maltose binding protein(MBP) was constructed via homologous recombination. A fragment of humanzalpha11 cDNA (SEQ ID NO:7) was isolated using PCR. The polynucleotidesequence for the MBP-zalpha11 soluble receptor fusion polypeptide isshown in SEQ ID NO:96. Two primers were used in the production of thehuman zalpha11 fragment in a PCR reaction: (1) Primer ZC20,187 (SEQ IDNO:98), containing 40 bp of the vector flanking sequence and 25 bpcorresponding to the amino terminus of the human zalpha11, and (2)primer ZC20,185 (SEQ ID NO:99), containing 40 bp of the 3′ endcorresponding to the flanking vector sequence and 25 bp corresponding tothe carboxyl terminus of the human zalpha11. The PCR Reaction conditionswere as follows: 25 cycles of 94° C. for 30 seconds, 50° C. for 30seconds, and 72° C. for 1 minute; followed by 4° C. soak, run induplicate. Two μl of the 100 μl PCR reaction was run on a 1.0% agarosegel with 1×TBE buffer for analysis, and the expected approximately 660bp fragment was seen. The remaining 90 μof PCR reaction was combinedwith the second PCR tube precipitated with 400 μl of absolute ethanol.The precipitated DNA used for recombining into the Smal cut recipientvector pTAP98 (Example 31) to produce the construct encoding theMBP-zalpha11 fusion. Clones were transformed, identified and grown up asdescribed in Example 31. The positive clones were designated pCZR225 andsubjected to sequence analysis. The polynucleotide sequence for theMBP-zalpha11 soluble receptor fusion polypeptide is shown in SEQ IDNO:96, and corresponding polypeptide sequence is shown in SEQ ID NO:97.The positive clones were used to grow up in E. coli as described inExample 31 for protein purification of the huzalpha11/MBP-6H fusionprotein (Example 46, below).

Example 46 Purification of Huzalpha11/MBP-6H Soluble Receptor From E.coli Fermentation

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying huzalpha11/MBP-6H solublereceptor polypeptide. E. coli cells containing the pCZR225 construct andexpressing huzalpha11/MBP-6H soluble receptor (Example 45) were grown upin SuperBroth II (12 g/L Casein, 24 g/L Yeast Extract, 11.4 g/Ldi-potassium phosphate, 1.7 g/L Mono-potassium phosphate; BectonDickenson, Cockeysville, Md.), and frozen in 0.5% glycerol. Twenty gramsof the frozen cells in SuperBroth II + Glycerol were used to purify theprotein. The frozen cells were thawed and diluted 1:10 in a proteaseinhibitor solution (Extraction buffer) prior to lysing the cells andreleasing the huzalpha11/MBP-6H soluble receptor protein. The dilutedcells contained final concentrations of 20 mM Tris (JT Baker,Philipsburg, N.J.) 100 mM Sodium Chloride (NaCl, Mallinkrodt, Paris,Ky.), 0.5 mM pheynlmethylsulfonyl fluoride (PMSF, Sigma Chemical Co.,St. Louis, Mo.), 2 μg/ml Leupeptin (Fluka, Switzerland), and 2 μg/mlAprotinin (Sigma). A French Press cell breaking system (Constant SystemsLtd., Warwick, UK) with temperature of −7 to −10° C. and 30K PSI wasused to lyse the cells. The diluted cells were checked for breakage byA₆₀₀ readings before and after the French Press. The lysed cells werecentrifuged at 18,000 G for 45 minutes to remove the broken cell debris,and the supernatant used to purify the protein. Total target proteinconcentrations of the supernatant was determined via BCA Protein Assay(Pierce, Rockford, Ill.), according to manufacturer's instructions.

A 25 ml column of Talon Metal Affinity resin (Clontech, Palo Alto,Calif.) (prepared as described below) was poured in a Bio-Rad, 2.5 cmD×10 cm H glass column. The column was packed and equilibrated bygravity with 10 column volumes (CVs) of Talon Equilibration buffer (20mM Tris, 100 mM NaCl, pH 8.0). The supernatant was batch loaded to Talonmetal affinity resin and was rocked overnight. The resin was poured backinto the column and was washed with 10 CV's of Talon Equilibrationbuffer by gravity, then gravity eluted with 140 ml of Elution buffer(Talon Equilibration buffer+200 mM Imidazole-Fluka Chemical). The taloncolumn was cleaned with 5 CVs of 20 mM 2-(N-Morhpholino) ethanesulfonicacid pH 5.0 (MES, Sigma), 5 CVs of distilled H₂O, then stored in 20%Ethanol/0.1% Sodium Azide. Fourteen ml fractions were collected over theentire elution chromatography and the fractions were read withabsorbance at 280 and 320 nM and BCA protein assay; the pass through andwash pools were also saved and analyzed. The protein elution fractionsof interest were pooled and loaded straight to Amylose resin (NewEngland Biolabs, Beverly, Mass.).

To obtain more pure huzalpha11/MBP-6H polypeptide, the talon affinityelution pooled fractions were subjected to Amylose resin (22 mls) at pH7.4. A 2.5 cm D×10 cm H Bio-Rad column was poured, packed andequilibrated in 10 CVs of Amylose equilibration buffer-20 mM Tris (JTBaker), 100 mM NaCl (Mallinkrodt), 1 mM PMSF (Sigma), 10 mMbeta-Mercaptoethanol (BME, ICN Biomedicals Inc., Aurora, Ohio) pH 7.4.The sample was loaded by gravity flow rate of 0.5 ml/min. The column waswashed for 10 CVs with Amylose equilibration buffer, then eluted withabout 2 CV of Amylose equilibration buffer+10 mM Maltose (FlukaBiochemical, Switzerland) by gravity. 5 ml fractions were collected overthe entire chromatography and absorbance at 280 and 320 nM were read.The Amylose column was regenerated with 1 CV of distilled H₂O, 5 CVs of0.1% (w/v) SDS (Sigma), 5 CVs of distilled H₂O, and then 5 CVs ofAmylose equilibration buffer.

Fractions of interest were pooled and dialyzed in a Slide-A-Lyzer(Pierce) with 4×4 L PBS pH 7.4 (Sigma) to remove low molecular weightcontaminants, buffer exchange and desalt. After the changes of PBS, thematerial harvested represented the purified huzalpha11/MBP-6Hpolypeptide. The purified huzalpha11/MBP-6H polypeptide was analyzed viaSDS-PAGE Coomassie staining and Western blot analysis with theanti-rabbit HRP conjugated antibody (Rockland, Gilbertsville, Pa.). Theconcentration of the huzalpha11/MBP-6H polypeptide was 1.92 mg/ml asdetermined by BCA analysis.

Purified huzalpha11/MBP-6H polypeptide was prepared for injection intorabbits and sent to R & R Research and Development (Stanwood, Wash.) forantibody production. Rabbits were injected to produce antianti-huzalpha11/MBP-6H serum (Example 47, below).

Example 47 Zalpha11 Receptor Polyclonal Antibodies

Polyclonal antibodies were prepared by immunizing two female New Zealandwhite rabbits with the purified huzalpha11/MBP-6H polypeptide (Example46), or the purified recombinant zalpha11CEE soluble receptor (Example10A). Corresponding polyclonal antibodies were designated rabbitanti-huzalpha11/MBP-6H and rabbit anti-huzalpha11-CEE-BHK respectively.The rabbits were each given an initial intraperitoneal (IP) injection of200 mg of purified protein in Complete Freund's Adjuvant (Pierce,Rockford, Ill.) followed by booster IP injections of 100 mg purifiedprotein in Incomplete Freund's Adjuvant every three weeks. Seven to tendays after the administration of the third booster injection, theanimals were bled and the serum was collected. The rabbits were thenboosted and bled every three weeks.

The zalpha11-specific polyclonal antibodies were affinity purified fromthe rabbit serum using an CNBr-SEPHAROSE 4B protein column (PharmaciaLKB) that was prepared using 10 mg of the purified huzalpha11/MBP-6Hpolypeptide (Example 32) per gram CNBr-SEPHAROSE, followed by 20×dialysis in PBS overnight. Zalpha11-specific antibodies werecharacterized by an ELISA titer check using 1 mg/ml of the appropriateprotein antigen as an antibody target. The lower limit of detection(LLD) of the rabbit anti-huzalpha11/MBP-6H affinity purified antibody isa dilution of 500 pg/ml. The LLD of the rabbit anti-huzalpha11-CEE-BHKaffinity purified antibody is a dilution of 50 pg/ml.

Example 48 Zalpha11 Receptor Distribution

To assess zalpha11 receptor distribution on various cells types, wegenerated both rabbit polyclonal and mouse monoclonal antibodies (mAbs)directed against the human receptor (Example 35 and Example 47) andconjugated these antibodies to biotin for use in flow cytometry. Weinitially used the polyclonal antibodies, which were of relatively lowaffinity, to stain a panel of cell lines: L-3 dependent murine pre-Bcell line wild-type BaF3 cells (Palacios and Steinmetz, ibid.;Mathey-Prevot et al., ibid.); BaF3 cells transfected with human zalpha11(Example 4); human Burkitt's lymphoma cell lines Raji (ATCC No. CCL-86),Ramos (ATCC No. CRL-1596), RPMI 8226 (ATCC No. CCL-155), and Daudi (ATCCNo. CCL-213); human T cell leukemia cell line Jurkat (ATCC No. TIB-152);human myelomonocytic leukemia cell lines Thp-1 (ATCC No. TIB-202) andUT937 (ATCC No.CRL-1593.2); human pro-myelomonocytic cells HLT60 (ATCCNo. CCL-240); murine B cell lymphoma cell line A20 (ATCC No TIB-208);and murine thymoma cell line EL4 (ATCC No. TIB-39).

The cells were harvested, washed once with FACS wash buffer with serum(WBS). WBS consisted of Hank's balanced salt solution (Gibco/BRL)+10 mMHEPES (Gibco/BRL)+1% BSA (Sigma)+10% normal goat serum (GeminiBioproducts, Woodland, Calif.)+10% normal rabbit serum (Sigma). Washbuffer (WB) was identical to WBS except that it was serum free. Afterwashing, the cells were resuspended in 100 μl WB containing 10 μg/mlrabbit anti-zalpha11 polyclonal antibodies (Example 47). The cells werekept on ice with Ab for 20 min, then washed with WB and resuspended inWB containing goat anti-rabbit-FITC (BioSource, International),incubated another 20 min on ice, then washed and resuspended in 400 μlWB for analysis on a FACSCalibur flow cytometer (Becton Dickinson).Control samples were stained with the secondary goat anti-rabbit-FITC Abonly. Positive staining was defined as a shift above the staining withsecondary alone. Although the polyclonal antibodies were of lowaffinity, we confidently detected zalpha11 expression on theBaF3/zalpha11 transfectant, on all four human Burkitt's lymphomas (Raji,Ramos, Daudi, and RPMI 8226), and on Jurkat T cells. Resting(undifferentiated) HL-60 cells did not bind the anti-zalpha11antibodies, but we did detect a positive signal on HL-60 cells activatedfor 24 hours with PMA (Calbiochem, La Jolla, Calif.) which induces HL-60cell differentiation into a monocyte-like cell. We also saw a positivesignal on UT937 and Thp-1 cells, although this signal may have been dueto non-specific binding. The polyclonal antibodies weakly cross-reactedon the mouse B cell line A20, but we saw no staining of the EL4 murinethymoma.

The four anti-zalpha11 monoclonal antibodies (Example 35) wereconjugated to biotin, and a subset of the cells described above werescreened for zalpha11 receptor expression (BaF3, BaF3/zalpha11, Raji,Jurkat, and resting HL-60). Cells were harvested, washed, thenresuspended in 100 μl WB containing 15 μg/ml of one of each of the 4biotinylated mAbs. The cells were incubated with mAb for 20 min on ice,then washed with 1.5 ml WB and pelleted in a centrifuge. The supernatantwas removed by aspiration and the pellets were resuspended in 100 μl ofCyChrome-conjugated streptavidin (CyC—SA; PharMingen), then incubated onice for another 20 min and washed and pelleted as before. Control tubescontained cells stained only with CyC—SA. Pellets were resuspended in400 μl WB and flow cytometry performed as above. Positive staining wasdefined as a signal exceeding the background level of staining withCyC—SA alone. Using the BaF3/zalpha11 transfectant as a control, we wereable to rankthe 4 mAbs in terms of their respective mean fluorescenceintensities (MFI), which can reflect antibody affinity and/or the extentof biotinylation of the mAbs. The mAbs were ranked as follows, fromhighest to lowest MFI: 249.28.2.1.2.2, 247.10.2.15.4.6, 249.19.2.2.3.5,and 249.15.2.4.2.7. The Raji cells stained positive with the zalpha11monoclonal antibodies. The Jurkats cells positively stained with thezalpha11 monoclonal antibodies, but not as strongly as that on B cells(Raji). Thus the, zalpha11 receptor was expressed on these B and T celllines. The staining patterns on non-activated HL60 cells were identicalfor all the mAbs, and the signal was very weak. We believe that thissignal does not reflect actual expression of zalpha11 by HL-60 cells,but rather may be due to non-specific binding of the mouse mAbs to thehuman cells, probably via Fc-receptors.

Example 49 Human Zalpha11 Ligand Effect on B-cells and Zalpha11 LigandToxic Saporin Fusion

The effects of human zalpha11 Ligand were tested on the following humanB-cell lines: and human Burkitt's lymphoma cell lines Raji (ATCCNo.CCL-86), and Ramos (ATCC No. CRL-1596); human EBV B-cell lymphomacell line RPMI 1788 (ATCC No. CRL-156); human myeloma/plasmacytoma cellline IM-9 (ATCC No. CRL159); and human EBV transformed B-cell lineDAKIKI (ATCC No. TIB-206), and HS Sultan cells (ATCC No. CRL-1484 ).Following about 2-5 days treatment with zalpha11 Ligand, changes insurface marker expression were found in IM-9, Raji, Ramos, and RPMI1788cell lines, showing that these cells can respond to zalpha11 Ligand.Human B-cell lines treated with zalpha11 Ligand grew much more slowlythan untreated cells when re-plated in cell culture dishes. These cellsalso had an increased expression of FAS ligand, as assessed by flowcytometry (Example 49D and Example 49E), and moderately increasedsensitivity to an activating FAS antibody (Example 49A). This resultsindicate that zalpha11 Ligand could control some types of B-cellneoplasms by inducing them to differentiate to a less proliferative andor more FAS ligand sensitive state. Moreover, zalpha11 receptor isexpressed on the surface of several of these cell lines (See Example48). Thus, zalpha11 Ligand and the human zalpha11 Ligand-saporinimmunotoxin conjugate (Example 49B, below), or other zalpha11Ligand-toxin fusion could be therapeutically used in B-cell leukemiasand lymphomas.

A. The Effect of Human Zalpha11 Ligand on B-Cell Lines

IM-9 cells were seeded at about 50,000 cells per ml±50 μg/ml purifiedhuman zalpha11 Ligand (Example 29). After 3 days growth the cells wereharvested, washed and counted then re-plated at about 2500 cells/ml in96 well plates in to wells with 0, 0.033, 0.1 or 0.33 μg/ml anti-FASantibody (R&D Systems, Minneapolis). After 2 days an Alamar bluefluorescence assay was performed (Example 2B) to assess proliferation ofthe cells.

Zalpha11 Ligand-treated IM-9 cells grew to only 27% the density of theuntreated cells in the absence of anti-FAS antibody. In the presence of0.33 μg/ml anti-FAS antibody, the zalpha11 Ligand-treated cells wereinhibited an additional 52% while the untreated cells were inhibited byonly 30%. The overall inhibition of cell growth with both zalpha11Ligand and 0.33 μg/ml anti-FAS antibody treatment was 86%.

When the IM-9 cells were pretreated for three days with or withoutzalpha11 Ligand and then re-plated at 100 cells per well and grown withor without anti-FAS antibody for 6 days, the growth of untreated cellsassessed by Alamar Blue assay (Example 2B) was inhibited only 25% byanti-FAS antibody while the growth of zalpha11 Ligand-treated cells wasinhibited 95% relative to the growth of untreated cells in zero anti-FASantibody.

B. The Effect of Human Zalpha11 Ligand-Saporin Immunotoxin on B-CellLines

The human zalpha11 Ligand-saporin immunotoxin conjugate (zalpha11L-sap)construction and purification is described in Example 50. The humanzalpha11L-sap was far more potent than the saporin alone in inhibitingcell growth. When the treated cell are re-plated after a three or fourday treatment the human zalpha11L-sap treated cells grew very poorly.

IM-9, Ramos and K562 (ATCC No. CCL-243) cells were seeded at about 2500cells/well in 96 well plates with zero to 250 ng/ml human zalpha11L-sapconjugate or 0-250 ng/ml saporin (Stirpe et al., Biotechnology10,:405-412, 1992) only as a control. The plates were incubated 4 daysthen an Alamar Blue proliferation assay was performed (Example 5B). Atthe maximal concentration of human zalpha11-sap conjugate, the growth ofIM-9 cells and RAMOS cells was inhibited by 79% and 65% respectively.K562 cells which are low/negative by flow for expression of the zalpha11receptor were not affected by the zalpha11-sap, thus showing thespecificity of the conjugate's effect.

IM-9 cells were seeded a 50,000 cells/ml into 6 well plates at zero and50 ng/ml human zalpha11L-sap conjugate. After 3 days the cells wereharvested and counted then re-plated from 100 to 0.8 cells per well in 2fold serial dilutions, and 12 wells per cell dilution without the humanzalpha11 Ligand-saporin immunotoxin. After 6 days the number of wellswith growth at each cell dilution was scored according to the results ofan Alamar blue proliferation assay (Example 2B).

When cell number was assessed, by Alamar blue assay (Example 2B), after6 days of growth control cells seeded at about 12.5 and 6.25 cells perwell had equivalent growth to zalpha11 -sap treated cells seeded at 100and 50 cells/well respectively. Thus, the growth of the survivingtreated IM-9 cells was markedly impaired even after the removal, byre-plating, of the zalpha11-sap immunotoxin.

The limited tissue distribution of the human zalpha11 receptor (Example48), and the specificity of action of the zalpha11-sap toreceptor-expressing cell lines suggest that this conjugate may betolerated in vivo.

C. The Effect of Human Zalpha11 Ligand-Saporin Immunotoxin on B-CellLine Viability

HS Sultan cells (ATCC No. CRL-1484 ) were seeded at about 40,000 cellsper ml into 12 well plates and grown for five days with either no addedcytokines or 4 0 ng/ml purified human zalpha11 Ligand (Example 29) or 25ng/ml human zalpha11L-sap conjugate (Example 50, below) or with 20 ng/mlIFN-alpha (RDI) or zalpha11 Ligand and IFN-alpha. Zalpha11 ligandinhibited the outgrowth of Hs Sultan cells by 63%. IFN-alpha inhibitedthe growth by 38%. Zalpha11 ligand plus IFN-alpha inhibited growth 78%,indicating that the growth inhibitory effects of human zalpha11 Ligandand IFN-alpha may be additive. The human zalpha11L-sap inhibited growthof the HS Sultans by 92%.

The results above support the possible use of zalpha11 Ligand or humanzalpha11L-sap in the treatment of malignancies or other diseases thatexpress the zalpha11receptor, particularly those of B-cell origin. Thecombination of zalpha11 Ligand with IFN-alpha is specifically suggestedby their additive effect in the inhibition of HS Sultan cells. Someother types of lymphoid malignancies and diseases may also express thezalpha11 receptor, as activated T-cells also express the receptor mRNA(Example 48), and some of these diseases may also be responsive tozalpha11 Ligand of zalpha11 Ligand-toxic fusion therapy.

D. FAS (CD95) Expression on Human B-Cell Lines is Increased by HumanZalpha11 Ligand Stimulation

Human B-cell lines HS Sultan (ATCC No. CRL-1484), IM-9 (ATCC No.CRL159), RPMI 8226 (ATCC No. CCL-155), RAMOS (ATCC No. CRL-1596), DAKIKI(ATCC No. TIB-206), and RPMI 1788 (ATCC No. CRL-156), were all treatedwith or without purified 10 to 50 ng/ml human zalpha11 Ligand (Example29) for 2 to 8 days. The cells were then stained with anti-CD95PE-conjugated antibody (PharMingen, San Diego, Calif.), permanufacture's protocol, and analyzed on a FACScalibur (Becton Dickinson,San Jose, Calif.). In all cell lines, anti-CD95 (FAS or APO-1) stainingwas increased, in some cases more than two fold, upon treatment withhuman zalpha11 Ligand.

E. FAS (CD95) Expression on Primary Mouse Spleen B-Cells is Increased byHuman Zalpha11 Ligand Stimulation

Primary mouse splenocytes were obtained by chopping up spleens from 8 to12 week old C57/BL6 mice. Erythrocytes were lysed by treating thepreparation for 5 seconds with water then put through a 70 micron sieve.The remaining splenocytes were washed and plated in RPMI (JRHBioscience) plus 10% HIA-FBS (Hyclone, Logan, Utah). Interleukin 2(IL-2) (R and D Systems) with or without human zalpha11 Ligand, asdescribed above. They were then incubated at 37° C., in 5% CO₂ for 5days. The splenocytes were harvested and stained with anti-CD95 PEconjugated antibody (PharMingen) and anti-CD19 FITC conjugated antibody(PharMingen) per manufacturer's protocol. The cells were analyzed byflow cytometry on a FACScalibur (Becton Dickinson). Upon gating on theCD19+ mouse B-cells, it was found that anti-CD95 staining was increasedon B-cells treated with IL-2 plus human zalpha11 Ligand compared tothose in IL-2 alone. The anti-CD95 staining was 37 relative fluorescentunits (RFU) on the B-cells in IL-2 alone and 55 RFU on the B-cellscultured in IL-2 and human zalpha11 Ligand.

Example 50 Construction and Purification of Zalpha11 Ligand Toxic Fusion

Under a supply contract, 10 mg human zlpha11 Ligand (Example 29) wassent to Advanced Targeting Systems (ATS, SanDiego, Calif.) forconjugation to the plant toxin saporin (Stirpe et al., Biotechnology10,:405-412, 1992). Zymogenetics received from ATS 1.3 mg of a proteinconjugate comprised of 1.1 molecules saporin per molecule of humanzlpha11 Ligand, formulated at a concentration of 1.14 mg/ml in 20 nMSodium phosphate, 300 nM sodium cloride, pH 7.2.

Example 51 Zalpha11 Ligand Toxic Fusion In Vivo

A. Testing Zalpha11-Saporin Conjugate in Mice

Zalpha11-saporin conjugate (Example 49) was administered to C57BL6 mice(female, 12 weeks of age, purchased from Taconic) at two differentdosages: 0.5 and 0.05 mg/kg. Injections were given i.v. in vehicleconsisting of 0.1% BSA (ICN, Costa Mesa, Calif.). Three injections weregiven over a period of one week (day 0, 2, and 7). Blood samples weretaken from the mice on day 0 (pre-injection) and on days 2 and 8(post-injection). Blood was collected into heparinized tubes (BectinDickenson, Franklin Lakes, N.J.), and cell counts were determined usingan automated hematology analyzer (Abbot Cell-Dyn model No. CD-3500CS,Abbot Park, Ill.). Animals were euthanized and necropsied on day 8following blood collection. Spleen, thymus, liver, kidney and bonemarrow were collected for histopathology. Spleen and thymus wereweighed, and and additional blood sample was collected in serumseparator tubes. Serum was sent to Pheonix Central Labs, Everett, Wash.,for testing in a standard chemistry panel. Samples were also collectedfor flow cytometric analysis as described herein.

Circulating blood cell counts and serum chemistry measurements did notdiffer significantly between zalpha11 conjugate treated mice and micetreated with an equivalent dose of unconjugated toxin (saporin).Histological analysis of tissues in zalpha11 -saporin treated miceshowed no significant changes relative to mice treated with anequivalent dose of unconjugated toxin. These results indicated that thesaporin conjugate was not toxic in vivo.

B. Testing Zalpha11 Ligand Toxic Saporin Fusion on B-Cell Derived TumorsIn Vivo

The effects of human zalpha11 Ligand and the human zalpha11 Ligand toxicsaporin fusion (Example 50) on human tumor cells are tested in vivousing a mouse tumor xenograft model described herein. The xenograftmodels are initially tested using cell lines selected on the basis of invitro experiments, such as those described in Example 49. These celllines include, but are not limited to: human Burkitt's lymphoma celllines Raji (ATCC No.CCL-86), and Ramos (ATCC No. CRL-1596); human cellline RPMI 1788 (ATCC No. CRL-156); human myeloma/plasmacytoma cell lineIM-9 (ATCC No. CRL159); human cell line DAKIKI (ATCC No. TIB-206), andHS Sultan cells (ATCC No. CRL-1484). Cells derived directly from humantumors can also be used in this type of model. In this way, screening ofpatient samples for sensitivity to treatment with zalpha11 Ligand orwith a zalpha11 Ligand toxic saporin fusion can be used to selectoptimal indications for use of zalpha11 in anti-tumor therapy.

After selection of the appropriate zenograft in vivo model, describedabove, zalpha11 Ligand-induced activity of natural killer cells and/orzalpha11 Ligand effects on B-cell derived tumors are assessed in vivo.Human zalpha11 Ligand is tested for its ability to generate cytotoxiceffector cells (e.g. NK cells) with activity against B-cell derivedtumors using mouse tumor xenograft models described herein. Moreover,direct affects of human zalpha11 Ligand on tumors can be assessed. Thexenograft models to be carried out are selected as described above. Aprotocol using zalpha11 Ligand stimulated human cells is developed andtested for efficacy in depleting tumor cells and promoting survival inmice innoculated with cell lines or primary tumors.

Example 52 Identification of P1 Artificial Chromosome Clones ContainingGenomic Human Zalpha11 Ligand DNA

The human zalpha11 Ligand cDNA insert was amplified by PCR usingvector-based primers. The PCR product was ³²P-labeled and hybridized tohigh-density filters representing a PAC (P1 Artificial Chromosome)library. Filters and frozen library stocks were obtained from RoswellPark Cancer Institute, Buffalo, N.Y.; the library segment was RPCI6,with a four-fold depth of coverage. Filters were hybridized overnight at65° C. in ExpressHyb (Clontech) and were washed according tomanufacturer's suggestions. Decoding the positive signals resulted inidentification of four PAC clones, designated 23H17, 34A9, 105G9, and236H14. PCR analysis using primers specific for the 5′ end (ZC22,452(SEQ ID NO:100) and ZC 22,451 (SEQ ID NO: 101)) and 3′ end (ZC 22,450(SEQ ID NO: 102) and ZC 22,449 (SEQ ID NO:103)) of the coding regionshowed that PACs 34A9 and 105G9 contained both ends, while PACs 23H17and 236H14 contained the 5′ end only. PAC 23H17 was digested with Eco RIand Not I, and a 9 kb fragment was identified which hybridized with thezalpha11 Ligand cDNA probe. This fragment was isolated and subcloned,using methods described herein, into pBluescript II SK (+) (Stratagene)previously digested with Eco RI and Not I. Sequencing revealed that thisfragment contained about 380 base pairs of the promoter region, exons 1,2, and 3, all of introns 1 and 2, and ended within intron 3.

The 3′ end of the human zalpha11 Ligand gene was obtained by PCR usingDNA from PAC 34A9 as template, with primers ZC23,771 (SEQ ID NO:104) andZC22,449.(SEQ ID NO:103). Taq DNA polymerase was used, with bufferprovided, with the addition of 4% DMSO. Reaction conditions were asfollows: 94° C., 5 min.; followed by 35 cycles of 94° C. for 30 sec.,52° C. for 1 min., 72° C. for 3 min.; then 72° C. for 7 min. Thisgenerated a 2.9 kb fragment which contained part of exon 3, all ofintrons 3 and 4, all of exon 4, and the coding portion of exon 5.

The genomic structure of the human zalpha11 Ligand gene is as followsfrom 5′ to 3′: SEQ ID NO:105, containing about 240 bp of the promoter,exon 1 (nucleotide number 240-455 of SEQ ID NO: 105), intron 1(nucleotide number 456-562 of SEQ ID NO: 105), exon 2 (nucleotide number563-598 of SEQ ID NO: 105), and part of intron 2 containing the 5′ 748base pairs (nucleotide number 599-1347 of SEQ ID NO: 105); a gap ofapproximately 3 kb; SEQ ID NO: 106, containing the 3′ 718 bp of intron2, exon 3 (nucleotide number 719-874 of SEQ ID NO: 106), and part of the5′ end of intron 3 (nucleotide number 875-1656 of SEQ ID NO: 106); a gapof less than about 800 bp; SEQ ID NO: 107, containing 644 bp of intron3; a gap of less than about 800 bp; and SEQ ID NO:108, containing the 3′435 bp of intron 3, exon 4 (nucleotide number 436-513 of SEQ ID NO:108), intron 4 (nucleotide number 514-603 of SEQ ID NO:108), and part ofthe 5′ end of exon 5 (nucleotide number 604-645 of SEQ ID NO: 108).

Example 53 ¹²⁵I-Labeled Human Zalpha11 Ligand Binding Study in CellLines

25 micrograms of purified human zalpha11 Ligand (Example 29) was labeledwith 2 mCl ¹²⁵I using iodobeads (Pierce, Rockford Ill.), according tomanufacturer's instructions. This labeled protein was used to asseshuman zalpha11 Ligand binding to human Raji cells (ATCC No. CCL-86),using binding to wild-type murine BaF3 cells, and BaF3 cells transfectedwith zalpha11 receptor (BaF3/hzalpha11 cells) as controls. Zalpha11Ligand binding to BaF3/hzalpha11 cells was expected (positive control),while no binding to wild-type BaF3 cells was expected (negativecontrol), based on proliferation assay results (Example 5). About 5×10⁵Raji cells/well, 1×10⁶ BaF3/hzalpha11 and 1×⁶ BaF3 cells cells/well,were each plated in 96-well plates. Ten ng/ml of labeled human zalpha11Ligand was added in duplicate to wells, with a dilution series ofunlabeled human zalpha11 Ligand competitor added from 250 fold molarexcess in 1:4 dilutions down to 0.061 fold molar excess. Each point wasrun in duplicate. After the labeled human zalpha1 Ligand was added towells, it was allowed to incubate at 4° C. for 2 h to allow for bindingof Ligand to the cells. The cells were then washed 3× in binding buffer(RPMI-1710 (JRH Bioscience) with 1% BSA (Sigma)), and counted on theCOBRA II AUTO-GAMMA gamma counter (Packard Instrument Company, Meriden,Conn.).

Binding of the labeled zalpha11 Ligand to cells was evident in the Rajiand the BaF3/hzalpha11 cells. In addition, for Raji cells, an average250 fold molar excess of unlabeled zalpha11 Ligand decreased binding 3fold in the presence of a non-specific unlabeled competitor (InterferonGamma from R&D Systems, Minneapolis, Minn.), and 3.7 fold relative to nocompetitor. Competition was observed in a dose dependent fashion for thespecific unlabeled competitor, human zalpha11 Ligand. Thus, the zalpha11Ligand binding to Raji cells was specific. Similarly, for positivecontrol BaF3/zalpha11 cells, the 250 fold molar excess of unlabeledzalpha11 Ligand decreased binding 2 fold relative to the non-specificcompetitor and 3.06 fold relative to no competitor. Thus, the zalpha11Ligand binding to BaF3/zalpha11 cells also was specific. No compeatablebinding was observed with the wild-type BaF3 cells. Thus, the zalpha11Ligand was shown to bind specifically to Raji cells, and toBaf3/hzalpha11 cells, but not to the negative control Baf3 cells.

Example 54 Zalpha11 Receptor Expression On Human Blood Cells

A. Preparation and Culture of Human Peripheral Blood Cells

Fresh drawn human blood was diluted 1:1 with PBS (GIBCO BRL) and layeredover Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology Inc., Piscataway,N.J.) and spun for 30 minutes at 1800 rpm and allowed to stop with thebrake off. The interface layer was removed and transferred to a fresh 50ml Falcon tube (Falcon, VWR, Seattle, Wash.), brought up to a finalvolume of 40 ml with PBS and spun for 10 minutes at 1200 rpm with thebrake on. The viability of the isolated cells was tested using TrypanBlue (GIBCO BRL) and the cells were resuspended at a final concentrationof 1×10⁶ cells/ml cell medium (RPMI Medium 1640, 10% Heat inactivatedfetal bovine serum, 5% L-glutamine, 5% Pen/Strep) (GIBCO BRL).

Cells were cultured in 6 well plates (Falcon, VWR) for 0, 4 or 24 hourswith a variety of different stimuli described below. Anti-IgM, anti-CD40and anti-CD3 stimulation were done as in Example 44 and Example 42.Phorbol myristate acetate (PMA) and ionomycin (Sigma, St. Louis, Mo.)(Example 5C) were added to appropriate wells at 10 ng/ml and 0.5 mg/mlrespectively. The cells were incubated at 37° C. in a humidifiedincubator for various times.

B. Antibody Staining and Analysis

Cells were collected out of the plates, washed and resuspended in icecold staining media (HBSS, 1% fetal bovine serum, 0.1% sodium azide) ata concentration of about ten million cells per milliliter. Blocking ofFc receptor and non-specific binding of antibodies to the cells wasachieved by adding 10% normal goat serum (Gemini Bioproducts, Woodland,Calif.) and 10% normal human serum (Ultraserum, Gemini) to the cellsuspension. Aliquots of the cell suspensions were mixed with a FITClabeled monoclonal antibody against one of the lineage markers CD3, CD19or CD14 (PharMingen, La Jolla, Calif.) and a biotinylated monoclonalantibody against the human zalpha11 receptor (hu-zalpha11) (Example 35).Staining specificity was determined by competition using zalpha11CEEsoluble receptor (Example 10A) at a ten fold mass excess. Afterincubation on ice for 60 minutes the cells were washed twice with icecold staining media and resuspended in 50 ml staining media containingstreptavidin-PE (Caltag, Burlingame, Calif.). After a 30 minuteincubation on ice, the cells were washed twice with ice cold wash buffer(PBS, 1% fetal bovine serum, 0.1% sodium azide) and resuspended in washbuffer containing 1 mg/ml 7-AAD (Molecular Probes, Eugene, Oreg.) as aviability marker. Flow data was acquired on living cells using aFACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose,Calif.). Both acquisition and analysis were performed using CellQuestsoftware (BD Immunocytometry Systems).

Results of staining by anti-zalpha11 antibody showed that the humanzalpha11 receptor is expressed on human peripheral blood cellsexpressing either CD3, CD19 or CD14. Staining on CD3 and CD19 cells wasspecific, as evidenced by absolute competion with the zalpha11 solublereceptor. Staining on CD14 cells showed some specificity for the Ligand,as evidenced by partial competion with the soluble receptor. Activationof either T cells with anti-CD3 or B cells with anti-CD40 resulted in anincreased level of cell surface zalpha11 at 24 hours. No increase in thelevel of expression of zalpha11 was seen at 4 hours with any stimulus oneither cell population. Treatment of the cells with zalpha11 ligandresulted in a decrease of zalpha11 staining on CD3 positive and CD19positive cells but not CD14 positive cells at both 4 and 24 hours.

Example 55 Preliminary Evaluation of the Aqueous Stability of HumanZalpha11 Ligand

Preliminary studies were conducted to evaluate the aqueous stabilitycharacteristics of human zalpha11 Ligand in support of bioprocessing,formulation, and in vivo administration. The objectives were to: 1)verify the stability and recovery from Alzet Minipumps & general storageand handling, 2) determine the stability-indicating nature of severalanalytical methods including cation-exchange HPLC (CX—HPLC),reverse-phase HPLC (RP—HPLC), size exclusion HPLC (SEC—HPLC), & bioassay(BaF3/zalpha11R proliferation (e.g., Example 2 and Example 4), and 3)determine the stability-limiting degradation pathways and their kineticdependencies.

Aliquots of purified human zalpha11 Ligand (Example 29) were prepared bydilution to 2 mg/mL in PBS (pH 7.4) and stored in low densitypolyethylene (LDPE) cryovials (Nalgene, 1.8 mL) at −80° C. (control), 5°C., 30° C., and 37° C. Samples were assayed intermittently over 29 daysby CX—, RP—, SEC—HPLC, and bioassay. Aliquots were also stored at −80°C. and subjected to freeze-thaw (f/t) cycling (−80° C./RT; 5×f/t,10×f/t). Recovery of human zalpha11 Ligand was determined relative tothe −80° C. control (1 f/t) in all assays.

The remaining human zalpha11 Ligand solution from the −80° C. controlsamples were refrozen (−80° C.) after analysis. This aliquot (2 f/t) wasused to evaluate the thermal and conformational stability of humanzalpha11 Ligand as a function of pH using circular dichroism (CD). The 2mg/mL solution was diluted to 100 μg/mL in PBS buffers ranging from pH3.3-8.8. The far-UV CD spectra was monitored over the temperature range5-90° C. in 5° C. intervals (n=3 pH). The CD spectropolarimeter used wasa Jasco 715 (Jasco, Easton, Md.). The thermal unfolding was monitored bychanges in ellipticity at 222 nm as a function of temperature. Estimatesof the T_(m) were estimated assuming a two-state unfolding model. Thedata was fit (sigmoidal) using SlideWrite Plus for Windows v4.1(Advanced Graphics Software; Encinitas, Calif.).

Recovery and stability from Alzet Minipumps (Model No. 1007D; ALZACorporation, Mountain View, Calif.) was assessed by filling pumps with100 μL of the 2 mg/mL human zalpha11 Ligand solution, placing the pumpsin 1.8 mL LDPE containing 1 mL of PBS (pH 7.4), and storing them at 37°C. The release/recovery of human zalpha11 Ligand from the minipumps wasassessed by CX—, RP—, and SEC— HPLC on days 2, 4, and 7. The activitywas assessed by bioassay on day 7. The study was designed to evaluatethe release from 3 pumps per sampling time.

The chromatographic data suggested that the CX— & SEC—HPLC methods werestability-indicating, whereas the RP—HPLC method was not. At least 3additional peaks indicating apparent degradation products were observedby CX—HPLC. The SEC—HPLC method resolved an apparent human zalpha11Ligand aggregate, eluting prior to human zalpha11 Ligand. However, nosignificant additional peaks were observed eluting after the humanzalpha11 Ligand peak. This suggests that the degradation productsobserved by CX—HPLC most probably result from amino acid modificationssuch as deamidation, rather than hydrolysis/proteolysis processesleading to clipped variants. A small degree of fronting/tailing wasobserved by RP—HPLC (relative to control) in samples which had beenshown to have undergone significant degradation by SEC— & CX—HPLC.However, apparent degradation products were not resolved by RP—HPLC. Thedegradation observed by CX—HPLC increased as a function oftime-temperature, and followed apparent first-order kinetics. The %human zalpha11 Ligand recovered by CX—HPLC after 29 days at 37° C., 30°C., and 5° C. was 39%, 63%, and 98%, respectively. Aggregation alsoincreased in a time-temperature dependent fashion. The % aggregate foundin preparations stored for 29 days at 37° C., 30° C., and 5° C. was 7.4,3.4, and below detectable limits (BDL), respectively. No significantdifferences were observed by bioassay in any sample, suggesting thedegradation products have equivalent activity to intact human zalpha11Ligand. No degradation was observed by any assay in samples subjected toup to 10 f/t cycles.

The release of human zalpha11 Ligand from Alzet Minipumps was consistentwith the theoretical expected volume release. This suggests thatsignificant surface adsorption would not impair the delivery of humanzalpha11 Ligand using the Alzet Minipumps with a 2 mg/mL fillconcentration. The degradation consistent with that previously noted wasobserved. The % purity determined by CX—HPLC of human zalpha11 Ligandreleased after 2, 4, and 7 days was 96%, 90%, and 79%, repectively. Itshould be recognized that degradation also occurs after human zalpha11Ligand is released into or diluted with release medium. Therefore, the %purity within the minipump may be somewhat different than thatdetermined to be in the release medium. The bioactivity of each samplewas consistent with the expected amount of human zalpha11 Ligandreleased from the minipumps.

The human zalpha11 Ligand far-UV CD spectra, as expected, was consistentwith interleukins, such as IL-3 (J. Biochem., 23:352-360, 1991), IL-4(Biochemistry, 30:1259-1264, 1991), and IL-6 mutants (Biochemistry,35:11503-11511, 1996). Gross changes in the far-uv CD spectra as afunction of pH were not observed. Results showed that the pH of maximumthermal/conformational stability was=pH 7.4. Analysis of the unfoldingcurves were based on a two-state unfolding mechanism to allow comparisonof the thermal/conformational stability as a function of pH/composition.However, one or more intermediates may exist during the unfoldingprocess since the cooperativity was relatively low, based on theshallowness of the unfolding curve. Although studies were notspecifically designed to determine whether human zalpha11 Ligand refoldsfollowing thermal unfolding to 90° C., preliminary data suggests that atleast partial refolding occurs after the temperature of the sample iscooled back to 20° C.

These studies allow an analytical paradigm to be identified to evaluatethe purity and verify the stability of human zalpha11 Ligand. Forinstance, SEC—HPLC can be used to characterize the extent and rate ofaggregation in aqueous solution. Likewise, CX—HPLC can be used tocharacterize the extent and rate of degradation of human zalpha11 Ligandby mechanisms other than aggregation. The bioassay can be used to verifyactivity of human zalpha11 Ligand and it's aqueous degradation products.For instance, the human zalpha11 Ligand variants generated in aqueoussolution & resolved by CX—HPLC may themselves be useful as therapeuticagents, since they have equivalent bioactivity. Also, the fact thathuman zalpha11 Ligand degrades by several different processes(aggregation, amino acid modifications) suggests a preferred or uniqueformulation which minimizes the rate of each degradation process may benecessary for long-term stability of a solution product.

Identification of the nature of the aqueous degradation products anddetermination of their kinetic dependencies (pH, concentration,excipients) is underway. Human zalpha11 Ligand stability in serum/plasmais determined to support the design and interpretation of in vivostudies.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A fusion protein comprising a first polypeptide comprising a sequenceof amino acid residues that is at least 90% identical to residues 41 to148 of SEQ ID NO: 2 and comprising a second lL-2 polypeptide as shown inSEQ 1D NO: 111 or fragment thereof, wherein the fusion protein binds thereceptor as shown in SEQ ID NO:
 115. 2. The fusion protein of claim 1,wherein the first polypeptide of at least 95% identical to residues 41to 148 of SEQ ID NO:
 2. 3. The fusion protein of claim 1, wherein thefirst polypeptide comprises residues 41 to 148 of SEQ ID NO:
 2. 4. Afusion protein comprising a first polypeptide comprising amino acidresidues 32 to 162 of SEQ ID NO: 2 and comprising a second lL-2polypeptide as shown in SEQ ID NO: 111 or fragment thereof, wherein thefusion protein binds the receptor as shown in SEQ ID NO: 115.